Measurement of Larval Activity in the Drosophila Activity Monitor

The overall goal of this procedure is to analyze larval activity without the use of complicated video analysis software. This is accomplished by first transferring selected larvae to a mesh filter to clean away debris. Next individual larvae are transferred to assay tubes where they are secured by auger plugs, which are inserted into a drosophila activity monitor or dam device.

After three to five minutes of acclimation recordings are taken from the DAM system for analysis. Ultimately, the dam measured larval movements can be used to analyze different activity parameters for a given genotype. One advantage this technique has over existing methods is that it is automated and objective.

While being simple enough for routine activity measurements, many laboratories already have the necessary equipment that can be easily adapted for use on larvae. Demonstrating the procedure will be Aiden McFarlin, an undergraduate student from my laboratory. This protocol uses an M five multi-beam drosophila activity monitor device or a dam.

To set up the dam. First, connect the monitor to the power supply interface unit or PSIU using CAB six telephone cable. Then connect the PSIU to a power outlet.

A green light will indicate an appropriate connection. Third, connect the PSIU through A USB cable to a computer for data recording. Now install and open the DAM system MB one V six X program on the computer.

When the software is opened, it automatically starts recording data in the software control. For the dam set the desired recording interval. Choose preferences and then click above or below the reading interval option to select different timeframes in which data will be stored.

There are a variety of other parameters that can be set found under preferences and under output data type. Each parameter provides a unique analysis of the larval activity within the tube. It is suggested that all parameters are selected prior to data collection.

If you are unsure of which parameter you wish to measure For this assay, prepare a vial with foraging larvae For each genotype intended to be assay. Use a standard environment and fly food to prepare the assay tubes. Pour a 4%auger solution to a depth of 1.5 centimeters into a Petri dish.

This becomes the plug material for the assay tube, too dense to be burrowed into. Next, ensure that the tubes are clean and unobstructed by washing them out with hot water. Next, heat a squeeze bottle with 100 milliliters of water in a microwave for 10 to 15 seconds or until condensation appears.

Then invert the bottle and gently squeeze the warm humid air into the assay tube. When a thin layer of condensation appears on the wall of the tube, it is sufficiently humid. Next, remove the gel disc from the dish and put it over a mesh surface, which it will not tightly adhere to.

To collect the larvae, prepare a mesh filter by stretching silk screen grade nylon mesh over a funnel. Secure the mesh at the funnel neck with a rubber band, and to position the funnel in a beaker. Now scoop a spatula full of food containing foraging larvae from the culture bottle and transfer them to the mesh filter.

Wash the food off the larvae using room temperature, tap water, individual larvae can be collected off the mesh using a paintbrush. Transfer the larvae about 1.5 inches into a humid assay tube. Then seal the tube by plugging it with auger.

This is done by pressing one end of the tube into the gel twice and the other end. Once pressure forces one of the two plugs out at the opposite end, as the tubes are loaded, place them into the dam. Next, adjust each tube's position so that the animal cannot move beyond the range of the sensors.

Then secure the tubes to the device using a ring of putty. Record the LARVAS activity in a 20 degree Celsius incubator to prevent inaccurate readings that may occur due to shadows, fluorescent lights, or to temperature variations in the lab. In the incubator.

Use LED lighting not fluorescent during the recording. Before collecting data, allow the larvae to acclimate for five minutes. It can be useful to perform a trial without any animals to ensure that light conditions do not trigger the DAM sensors.

After five minutes, start the pre-configured DAM software to initiate the recording. The assay can go on for 30 minutes without additional condensation or food. It's essential that prior to recording the activity parameter intended for analysis has been selected under preferences.

If not selected, this activity will not be available for post hoc analysis. After the desired recording time has completed close the DAM software, the data is automatically saved under the dam system data folder. Transfer the data file to a separate folder so it can be processed later.

The assay tubes can be cleaned out with boiling water for reuse. To process the raw data into an understandable format, open the DAM file scan application and choose. Select the input data folder, then choose the data folder and the desired file and select the scan option.

Lastly, select the bin length to the desired reading period. This organizes the raw data into parameters set by the operator. Now select the output data type to analyze the desired motion setting.

If the bin length is greater than the selected system reading interval, then go under the extra readings menu and select the option two sum into bins. Next, save the file under an appropriate name in the same folder as the raw data was transferred into. Using a standard spreadsheet program, open the saved file.

Depending on the data type analyzed, the spreadsheet must be read differently. Typically, to measure moves or counts, the time period of the study will be recorded numerically while each individual reading tube will receive a designated letter. Normally columns K to Z represent the slots for assay tubes, and the rows represent the data points collected.

If, for example, moves were collected for 20 minutes, in one minute, intervals average the 20 rows to calculate an average number of moves per minute and other measurements. A temperature response study of third instar larvae of the W 1 1 1 8 strain was made using the monitoring device to detect differences in larva, locomotion. At seven different temperatures, each larvae was analyzed for 20 minutes.

Clearly, the larvae got significantly more active as the environment heated up. For comparison, a hypoactive mutant of the W 1 1 18 strain was tested called inactive. The analysis indicated that inactive larvae were significantly less mobile than a control to test the limits of detection of the assay.

A comparison of larvae at different stages was made while much smaller than third in star larvae. The activity of first and second in star larvae was indeed detectable and measurable looking into changes in activity with time in the device. It was found that the activity of third in star larvae in each minute of the 20 minute assay remained relatively consistent.

Once mastered, this protocol provides an accurate, simple, cost-effective method to evaluate basic parameters of larval activity under a variety of experimental conditions.