Drosophila Shallow Chamber

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The shallow Drosophila chamber, initially described by Simon et al. (2010), features an innovative design that confines flies to a limited vertical space, ensuring that all behavioral interactions occur within a single layer of individuals. This design reduces the likelihood of flies obscuring one another and encourages more flies to move toward the center of the chamber. Consequently, this increases interaction frequency and enhances the quality of data captured by digital video recordings.

A slippery glass ceiling restricts the time flies can cling before they fall to the floor. Each chamber can accommodate groups of up to 50 flies. Visual stimuli are provided by a 12×12-inch array of 850 nm IR LEDs mounted beneath the chambers for backlighting. There are two available chamber sizes: a 12.7 cm diameter chamber for larger groups of Drosophila and a 7 cm model suited for studies of courtship and aggression.

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Description

Specifications

13 cm diameter (Group Chamber)

7 cm Diameter (Courtship)

11 degree sloped Flooring

11 degree angled floor

Glass ceiling

Glass ceiling

LED multi light display

Multiplex light array

Removable Plug used for courtship assays and solid resource used for observations of aggression and the conventional chamber optional. Inquire for quote.

Removable plug used for courtship assays and solid resource used for observations of aggression and the conventional chamber available upon request. Inquire for quote.

Introduction

The Drosophila Shallow Chamber (DSC) is an essential tool for examining the locomotor behavior of individual Drosophila melanogaster flies, interactions between pairs, or complex social interactions within large groups. Over the years, studies on Drosophila have provided significant insights into the molecular mechanisms governing behavior and development. Despite differences in morphology and cellular features, Drosophila melanogaster shares many functionally analogous internal organ systems with vertebrates, including humans. Historically, the morphological differences limited the use of invertebrate organs in studying pathological mechanisms. However, advancements in genomic mapping have revealed that approximately 65% of human disease-causing genes have functional homologs in flies, which express proteins with similar functions to those in humans. This discovery has been pivotal in identifying molecular mechanisms underlying mutations in the human genome that lead to disease phenotypes, particularly those affecting the nervous system. Drosophila, with its smaller size and less complex nervous system compared to many vertebrates, serves as a model for studying various neurological diseases, such as neurodegeneration, epilepsy, dementia, stroke, traumatic brain injury, and brain tumors. These studies offer novel insights into pathogenic mechanisms and aid in developing therapeutic strategies for neurological disorders (Ugur et al., 2016).

While numerous genetic tools exist to study the relationships between genes, neurons, and behavior in flies, the development of tools for quantifying behavior has lagged, slowing progress in understanding these relationships. Drosophila behavior is often studied through digital recordings using machine vision methodologies that automatically track and measure behavioral phenotypes. The effectiveness of these methodologies depends heavily on the quality of raw data recorded from experimental chambers. Traditional chambers with vertical walls pose significant challenges for behavioral measurement and analysis. Vertical walls enable flies to walk up and along the ceiling, causing overlapping and blocking behaviors, which complicates individual tracking. This movement can obscure key identifiable features such as the position of wings or limbs, critical for analyzing specific behaviors. High ceilings allow ample room for flight and space for flies to cluster in the periphery, making tracking difficult with a single camera. To address these issues, intricate designs incorporating thermal barriers and water moats were developed (Simon et al., 2010). These designs often required wing clipping to prevent flight, potentially interfering with behaviors such as courtship and aggression. Simon et al. introduced a simplified experimental chamber (DSC) with a low ceiling and sloping walls, creating a shallow space that forces flies to form a monolayer, preventing overlap and appearance changes. This chamber complements various machine vision methodologies for behavioral measurement and analysis using an overhead camera.

The Drosophila Shallow Chamber is a versatile experimental tool featuring sloping walls rather than vertical ones with square corners. The sloping walls intersect with a paint-coated glass ceiling and are continuous with the chamber’s horizontal floor. The floor has holes for introducing food and water. The chamber is illuminated from above with fluorescent lights and from below with infrared LED lights. A camera positioned above the chamber records fly behavior.

Apparatus and Equipment

The DSC is designed with unique variations compared to conventional chambers with vertical walls extending from floor to ceiling. The chamber features a 3.5 mm high glass ceiling coated with silicone paint, while the rest of the chamber is constructed from transparent clear acrylic mounted on an aluminum base. The floor has a thickness of 5 mm. The chamber walls consist of two sections: a sigmoid sloping part starting from the ceiling at an 11-degree angle, transitioning into a straight section that merges seamlessly with the floor, creating a continuous smooth surface.

The diameter of the chamber varies based on the study’s purpose, with a 7 cm diameter for observing courtship behavior and a 13 cm diameter for studying the movement of individual flies or groups. To ensure uniform backlighting, material is removed from the underside of the chamber following the same curvature as the floor. The floor is equipped with multiple holes fitted with removable plugs, providing access for food and water. The chamber is encased by a cylinder with a lid made from translucent checkered black and white paper.

For illumination, the chamber utilizes an array of fluorescent lights for backlighting, supplemented by standard fluorescent room lights from above and a 12×12-inch array of 850 nm infrared LEDs mounted underneath. A single digital camera is centrally mounted above the chamber to record fly behavior.

Training Protocol

Flies are housed in standard 250 mL bottles at 25°C and 40% relative humidity, supplied with standard food medium. Fly stocks are maintained on a 16-hour light to 8-hour dark cycle with immediate transitions between the photoperiods. A day before testing, collect 50 flies from the culture bottles and house them in standard 10 mL Drosophila vials with food. On the morning of the test, place 25 male and 25 female flies together in a standard Drosophila vial containing only agar, 7 hours prior to testing. For courtship behavior tests, isolate virgin flies 7 hours after they emerge from the pupal stage. Place male flies individually and group 15 female flies together in a vial with standard food for 4-5 days before testing.

Clean the entire chamber with ethanol and allow it to dry for 15 minutes. Use a mouth pipette to transfer the flies into the chamber. Immobilize the flies by cooling them down to 4°C for sorting and counting. Tracking and recording of the trials can be conducted using a system such as the Noldus EthoVision XT. For courtship behavior studies, place a pair of virgin male and female flies into a 7 cm chamber and record their behavior for 20 minutes or until copulation begins. For locomotion studies, introduce either a single fly or a group of up to 50 flies into the chamber and record their behavior for 30 minutes to 1 hour.

Simon et al. designed the DSC as a versatile experimental chamber to enhance automated machine vision methodologies for studying fruit fly behavior. Their comparative study with conventional chambers underscored the DSC’s unique design features. The DSC confines flies to a shallow space, ensuring the formation of a monolayer of individuals, which prevents overlapping and blocking. This design reduces variability in appearance and encourages more flies to move toward the center of the chamber, thereby increasing their interactions.

Such chambers represent significant progress in aligning with the development of advanced genetic manipulation tools. The DSC enables the collection of higher quality data for quantifying complex behavioral phenotypes of Drosophila, which can be translated into insights for human behavioral neuroscience studies.

Bussell et al. explored female sexual receptivity, focusing on how females integrate external sensory inputs from males with internal signals from their own reproductive systems to activate motor neuronal networks that may lead to copulation. In virgin females, pausing is a key behavioral aspect, regulated by a specific subpopulation of neurons identified through a genome-wide neuronal RNAi screen. They discovered that the Abdominal-B (Abd-B) homeobox transcription factor is essential during neuronal development for high levels of virgin female receptivity.

Using the DSC, Bussell et al. observed courtship behavior between female flies with either silenced or synthetically activated Abd-B neurons and adult male flies. The results showed that females with silenced Abd-B neurons exhibited decreased receptivity due to reduced pausing, whereas females with synthetically activated Abd-B neurons demonstrated increased pausing. Given that Drosophila courtship behavior is innate, Drosophila serves as an excellent model for studying the neural networks involved in innate social behaviors observed across various species.

Data Analysis

Tracking and recording the flies can be achieved through machine vision methodologies using automated tracking and video systems like Noldus EthoVision XT. This system is capable of measuring various parameters including velocity, distance traveled, time spent moving versus stationary, orientation, position, foraging behavior, circadian rhythms, courtship behavior, and thigmotaxis in Drosophila. Additionally, Noldus Observer XT can be employed to collect, analyze, and present observational data, enhancing the study of fly behavior.

Strengths and Limitations

The DSC features a shallow volume that confines all behavioral interactions to a single layer, preventing flies from overlapping or obscuring one another. This design minimizes variability in appearance and discourages flies from clustering in corners or the periphery, encouraging more movement toward the center and facilitating greater interactions among the flies. Unlike other designs, the DSC eliminates the need to clip wings to prevent flying or reduce ceiling walking. The ceiling is coated with paint to create a slippery surface, causing flies to fall back to the floor when they attempt to cling to it. The chamber’s diameter and height can be adjusted to suit different experimental needs.

The DSC is unsuitable for studying flight behavior in flies due to its limited volume, which restricts flying. Different aspects of behavioral data analysis may necessitate the use of multiple machine vision methodologies or tools. Additionally, the low ceiling of the chamber can hinder copulation and pose challenges for studying courtship behaviors.

Summary

  • Drosophila Shallow Chamber is used to study locomotion in individual Drosophila melanogaster flies or complex social interactions between a pair or group of flies.
  • DSC is a simple experimental chamber with a low ceiling and sloping angled walls creating a small shallow space that forces the flies to form a monolayer of individuals.
  • The chamber complements a variety of machine vision methodologies for measuring and analysis of behavior using an overhead viewing camera angle.
  • Drosophila behavior studies can provide novel insights into pathogenic mechanisms and help in devising new therapeutic strategies for different neurological diseases.

References

Simon JC, Dickinson MH (2010). A new chamber for studying the behavior of Drosophila. PLoS One. 5(1), e8793. DOI: 10.1371/journal.pone.0008793

Bussell JJ, Yapici N, Zhang SX, Dickson BJ, Vosshall LB (2014). Abdominal-B neurons control Drosophila virgin female receptivity. Current Biology 24(14), 1584-1595. DOI: 10.1016/j.cub.2014.06.011

Ugur B, Chen K, Bellen HJ (2016). Drosophila tools and assays for the study of human diseases. Disease Models & Mechanisms 9(3), 235-44. DOI: 10.1242/dmm.023762

Additional information

Dimensions

13cm, 7cm

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