Conditions Affecting Social Space in Drosophila melanogaster

The overall goal of this procedure is to quantify one aspect of the social behavior of flies, their preferred social distance. This is accomplished by first assembling the social space chamber. Meanwhile, a group of flies is habituated to the environmentally controlled assay room by being transferred into a new vial there.

After two hours, the group of flies is transferred to the social space chamber. They're tapped down and then positioned vertically with a clamp stand. Once the flies settle, their positions are documented, and the distances between the flies is calculated from this photographed by software.

Ultimately, the social space assay quantifies a form of social behavior, which can be used to study the influential environmental factors and neurogenetics of this behavior. The main advantage of this technique to quantify social behavior over existing methods like computer assisted video tracking, is that it does not require sophisticated tools or methods nor large investments in time and training. This method can help answer key questions in the social behavior field.

We have used this method as a diagnostic tool to study the role of autologous genes thought to be involved in social behavior and other organisms such as candidate genes for autism spectrum disorder in humans. I first had the idea for this method when I noticed at UCLA that in our stockroom flies in their vials, did not have a random distribution, but formed aggregates with ripped intervals in between them. This procedure describes the assembly of the social space chambers to begin while wearing gloves.

Place a triangular spacer on a square pane, aligning their corners in one location, then place a second triangular spacer to mirror the first one. Next, put the two small rectangular spacers on top of the square pane along the two uncovered edges, thus the four spacers form a triangular arena. Then sandwich the spacers with another square pane and use four clips to secure the assembly.

Placed a clips at the four corners beginning with the triangular spacer corners to ensure proper assembly. Orient the chamber vertically with the bottom edge resting on the bench. The room temperature should also be the same as the fly rearing temperature and similar humidity flies used with this experiment should be housed in bottles under standard conditions one or two days before the experiment, collect and sex the flies under cold anesthesia transfer.

Experimental populations of 40 flies into new fly vials. Later, two hours before the experiment, transfer the flies to new vials and put them on the bench where the arenas will be loaded two hours later. Begin the experiment.

Remove the nearest clip and slide out the rectangular spacer to make a one centimeter gap between the spacers. The upper left corner of the chamber can be used to identify the fly cohort being tested with a label. Just don't cover any aspect of the test arena.

Now transfer the cohort to a new empty vial and aspirate the flies from the vial. Next, insert the tip of the aspirator into the gap and gently exhale with even force. The flies should all be ejected into the arena.

Immediately slide the rectangular spacer back into position and replace the clip. Pound three times to knock all the flies to the bottom of the arena. Start the timer and take the arena into the controlled chamber.

Attach the arena vertically to the flask. Clamp in the controlled chamber along the edge of the chamber, but not over the arena. Attach a ruler or a sticker of known length after the flies.

Settle around 30 minutes. Photo, document the entire arena with the ruler in view. This concludes the trial.

Three replicates should be run for each condition. To unload the arena, place the arena in the freezer for five to 10 minutes and dump the frozen flies in a fly morgue org. In image J, open all the photos needed for the analysis.

Using the ruler, create a scale from the centimeter marks under the analyze tab. Choose set, scale, and follow the prompts. But before applying the scale, be sure to select the global option.

Using the crop feature, adjust the images to include just the arena space. Then select the type option and choose eight bit to render the images in black and white. Next, remove the background noise using the threshold feature under the adjust option of the image tab.

Drag the sliders until the body of each fly is clearly seen without creating marks in the background. To remove non fly marks, capture them with rectangle tool and delete them. Now configure the measurement tool in the analyze tab.

Select area center of mass Centro and display label from the available options. Then choose the analyze particles feature under the analyze tab. This creates a numbered list of all the flies lies.

Make certain this tool is working accurately by comparing the numbered list to the original photo. Now select the list. Then go under the plugins tab and select the macros option.

Followed by selecting the macro called nearest neighbor list distances. Analyze these values in a spreadsheet or statistical package using the described protocol. The recently established wild type line Elwood was compared with the inbred lab strains, Canton S some, and organ social spacing varied between the fly strains suggesting a genetic component to the behavior.

While I was hunting this procedure, it's important to remember to maintain controlled environmental conditions prior to and throughout the experiment. However, once achieved the orientation, the size and the shape of the arena do not matter and will not affect the behavior as long as you keep them constant After its development. This technique paved the way for researchers in the field of behavioral neuroscience to explore the effect of in environmental factors on social space.

In Joseph La Melanogaster, After watching this video, you should have a good understanding of how to conduct the social space assay, beginning from the preparation of flies to the setup and use of the social space arena, as well as image analysis.