Measurement of Larval Activity in the Drosophila Activity Monitor

Summary

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Abstract

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Introduction

The use of Drosophila as a genetic tool has transformed scientific knowledge of biological systems. Drosophila larvae have been used in a variety of studies including nociception¹, development² and as a model for the study of human disease genes³. Drosophila activity encompasses a range of behaviors that vary under different conditions including temperatures², exposure to drugs⁴ and amongst different genotypes. Yet, despite the significant use of the larva as a model organism, a simple, standardized method to analyze larval activity has not been available. Presently, many larval locomotion studies employ sophisticated video analysis software⁵. While powerful, the complexity of such automated tools may discourage labs that are not already equipped to study locomotion from including analysis of informative activity parameters in their studies. In other current non-automated methods, such as the grid crawling analysis, motion is scored by a human observer, which introduces the possibility of subjectivity and limits throughput to one animal at a time^6-7. A similar study used a 5-lane crawling assay, which measured the time it took larvae to travel a certain linear distance⁸. In such non-automated assays, displacement is measured but this does not account for non-linear travel between the start and end points. As discussed below, the method described here accounts for more of the actual larval movement, is objective, easy to operate, and offers robust throughput.

To easily study larval activity behavior without the compromise of accuracy, efficiency, or cost, this method employs the TriKinetics Drosophila Activity Monitor (DAM), a device often used to study adult activity. Using one device to study both adult and larval activity is cost-effective, and allows direct comparison of motion by animals at these two life stages. The system, featuring the manufacturer’s highest level of resolution, makes use of 17 infrared detection beams per assay tube, which record larval activity when sensor beams are broken within the 16 individual tubes. The system then automatically saves recorded information to a computer, making it available for manipulation with basic graphing software. The data obtained represents the beams that were broken by individual larvae (which can be converted into a rate), movement when a larva stays within a detector beam and the position of the animals within the assay chambers during a recording period (allowing one to calculate position preference). The system is efficient and relatively simple to operate, and brings highly reproducible basic activity analysis within the reach of any laboratory studying Drosophila larvae.

To demonstrate the power of this assay, data are presented that show its use to verify differences in activity resulting from varying ambient temperatures, as well as through the comparison of a mutant previously described as hypoactive (iav¹)⁹ with a widely analyzed control (w¹¹¹⁸).

Protocol

1. Preparation of Larvae

1. To analyze a desired larva for locomotion or position preference, grow larvae under standard conditions to the desired age to assay¹⁰ using standard fly food¹¹.
2. Make a mesh filter by stretching silk-screen grade nylon mesh over a funnel. Secure the mesh at the funnel neck with rubber band. Place the funnel in a beaker.
3. To collect larvae for analysis, scoop a spatula-full of food containing foraging larvae from the culture bottle and wash with RT tap water over the mesh filter, collecting individual larvae directly from the mesh with a paintbrush¹⁰.

2. Preparation of Assay Tubes

1. To prepare to plug the assay tubes, carefully boil a 4% agar gel and pour into a petri dish to a depth of 1.5 cm. This will provide a plug for either side of the tube when animal is enclosed. The 4% solution is sufficiently dense to prevent larvae from penetrating the plugs.
2. Ensure that tubes are clean and clear prior to insertion of larva. If they are not, this may block movements from being recorded.
3. To ensure larvae have sufficient moisture for movement, prepare a squeeze bottle containing a small amount of hot water. Invert the bottle, carefully orient the outlet toward a sink and squeeze gently to expel water from the pickup tube.
4. Insert the bottle outlet into the assay tube. Squeeze the bottle to deliver water vapor into the assay tube until a thin layer of condensation appears on the wall of the tube. Video 1 shows a larva moving in a tube with an appropriate amount of moisture.
5. To seal the larva in the tube, remove the 1.5 cm thick agar gel from the petri dish and place over a mesh surface to allow airflow beneath the agar so that an agar plug can be inserted into the tube. Press one end of the assay tube into the gel twice to insert two plugs of gel into one end.
6. With a paintbrush, place one larva into the tube approximately 1.5 inches deep and seal the tube by pressing the end nearest the larva into the agar gel. The resulting pressure will force out the second plug of agar gel from the opposite end and seal the animal within the tube.
7. Place tubes in the MB5 Multi-Beam Drosophila Activity Monitor(DAM) device, and adjust the tubes’ positions so that the animals cannot move beyond the range of the sensors.
8. To ensure that assay tubes do not slide out of the device’s infrared reading frame during transport, hold the tube in place by fastening a ring of putty around each tube where it contacts the recording device.

3. Measuring Activity

1. Record activity in an incubator to prevent inaccurate readings that may occur due to shadows, fluorescent lights or to temperature variations in the lab. See Figure 5 for a suggested arrangement of the system.
2. Set an incubator to 20 °C (see Figure 1). To avoid false recordings due to interference from incandescent light sources during recording, turn off fluorescent incubator lights and use a separate LED light source while recording in incubator. Perform a trial without any animals to ensure that light conditions do not aberrantly trigger sensors. There should be no recorded movement data after this test.
3. Allow larvae to acclimate to the 20°C incubator settings for 5 min prior to start of assay.
4. To set the DAM System to desired recording intervals (e.g., 1 min), ensure DAM system recording software is downloaded to host computer¹² and open the DAM System file prior to connecting the recording chamber to the PSIU interface.
5. To set the desired recording frequency at which data will be saved, select preferences and then click above or below the reading interval option to select different time frames in which data will be stored. For example, select reading interval times from 1 sec to 1 hr.
6. To select varying parameter for recording data, choose preferences and then select the corresponding boxes under output data type (counts, moves, positions and dwell, see Table 1). Each parameter provides a unique analysis of the larval activity within the tube.
7. To begin recording, connect the monitor to the Power Supply Interface Unit (PSIU) using CAB6 telephone cable. Connect the PSIU to a power outlet. A green light will indicate appropriate connection.
8. Connect the PSIU through a Universal Serial Bus (USB) cable to a Macintosh or Windows PC for data recording. Open the DAM SystemMB1v6x program on the computer to automatically start data recording.
9. After the desired collection time is complete, select Quit, and then Quit now on the DAM activity screen; data will then be automatically saved under DAM System Data. Take this data file (e.g., monitor 1) and drag into a separate folder. This will store the raw data from the DAM software so that they can later be processed.

4. Preparing Data for Processing (DAM FileScan)

1. To process the raw data into an understandable format open the DAM FileScan application and select Select 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 will organize the raw data into parameters set by the operator and the program will report the data collected in that particular range.
2. Select the output data type to analyze the desired motion setting (monitor counts, moves, dwell). Under the extra readings menu, select sum into bin (this is if the bin length is greater than the selected system reading interval).
3. Name the file appropriately and save. The file will be stored in the same location as the folder from step 3.9.

5. Accessing Data for Analysis

1. In order to collect average moves, process the raw data generated from step 4. To generate a table in a spreadsheet program, open the file that was previously saved (step 4.3), and when the text import wizard window appears, select finish to open the data files in a spreadsheet format.
   Note: Depending on the data type analyzed, the spreadsheet will be read differently. Typically to measure monitor moves or counts, the time period of the study will be recorded numerically, while each individual reading tube will receive a designated letter. When viewing the data in the spreadsheet, columns K-Z represent the slots for assay tubes 1-16 respectively. The rows represent the data points collected.
2. For example, if moves were collected for 20 min in 1 min intervals, average the 20 rows to calculate an average number of moves/minute and other measurements.

Representative Results

Figure 1 shows the results from a temperature response study of control third instar larvae, w¹¹¹⁸, using the monitoring device to detect differences in larval locomotion at seven different temperatures. Larvae were washed and placed into the DAM activity device as described above, and placed in an incubator set to the desired temperature. The apparatus was then allowed to acclimate to the environment for 5 min before recording began. Each larva was individually analyzed for locomotion over a 20 min period and the average number of moves per minute was calculated for every animal and averaged for each set of 32 animals. Data were analyzed and graphed using a spreadsheet program. Larvae exhibited significantly increasing activity as temperature increased correspondingly from 5-35 °C in 5-degree increments, except for a break in this trend at 20 °C and 25 °C.

To verify that differences could be detected between a control and a mutant previously described as hypoactive, inactive larvae (iav¹) were tested. Data were analyzed as moves/minute for each of the 32 animals and an average was then calculated. As shown in Figure 2, the analysis indicates that inactive larvae were significantly less mobile than a control. While they are much smaller than third instar larvae, activity of first and second instar larvae was also measurable, as shown in Figure 3. Activity of third instar larvae in each minute of the 20 min assay was shown to remain relatively consistent throughout the period (Figure 4).

Figure 1. w¹¹¹⁸ larval locomotion was recorded at temperatures varying from 5 °C – 35 °C. Each column represents the average motion of 32 third instar animals with individual moves per minute averaged amongst the set. *All averages are significantly different from each other except 10 °C and 15 °C (p=0.116) (Different letters indicate a significant difference, Student t-test).

Figure 2. Third instar control larvae compared to inactive (iav¹) larvae at 20 °C. iav¹ larvae exhibit significantly less mobility compared to control.

Figure 3. Measurement of activity of first, second and third instar larvae over a 20 min recording interval at 20 °C. N=32 for each larval stage.

Figure 4. Histogram displaying average number of moves occurring in each minute of the 20 min recording interval. 32 third instar larvae were assayed at 20 °C. The red line represents the number of moves per minute averaged for the entire 20 min interval.

Figure 5. Diagram illustrating the Drosophila Activity Monitor set-up and its connectivity to the PSIU interface unit and a desktop computer. The inside of the incubator is depicted, but during recording the incubator is to be shut.

Description	Parameter
Records data each time a larva crosses a beam, and additional counts are recorded if the larva moves while within a single beam.	Counts
Records data only when a fly repositions between separate beams, it does not record movement within a beam.	Moves
Records the animals position at each second over a recording interval. This data reveals position preference as a function of time spent at each sensor over the course of an assay.	Dwell
This setting analyzes the animal’s general position within the tube, indicating which sensor the animal is triggering during movement.	Position
This setting determines the frequency at which data collected during a set time frame is saved to the desktop computer.	Recording Interval

Table 1. Description of measurement parameters (Counts, Moves, Dwell and Positions).

Video 1. DAM system assay tube prior to insertion into operating device. Moisture is provided by condensation from breath. This level is sufficient to maintain larval locomotion throughout the study period without causing the animal to float or swim.Please click here to view this video.

Discussion

Activity of Drosophila larvae is influenced by a variety of factors including genotype⁸, age¹³ and ambient temperature². Although powerful videographic methods capable of highly detailed analysis have been developed by those who study locomotion⁵, this level of detail may be superfluous for those who wish to determine basic parameters of activity. The method described here employs a device that is available in many laboratories, is easy to operate, generates highly reproducible results, and is manageable even for those whose primary research focus is not locomotion. The example results demonstrate that this assay can be used to detect significant changes in motility in larvae subjected to different temperatures (Figure 1) and of different genotypes (Figure 2).

When larvae were measured at temperatures ranging from 5 °C – 35 °C, their activity increased with temperature, except for a break in the trend between 20 °C and 25 °C (Figure 1). It has been shown by Ainsley and coworkers that foraging early third instar larvae prefer temperatures within +/- 2 °C of the typical 25 °C culture temperature. However, when larvae enter wandering mid-third instar phase they prefer somewhat cooler temperatures². That finding is consistent with the observation that locomotor activity for third-instar larvae is greater at 20 °C than 25 °C, suggesting that some portion of animals assayed were in the wandering stage and more active at the cooler temperatures, and less so at the normal culture temperature of 25 °C.

This method offers simplicity, objectivity and robust throughput, but there are limitations. The applications described above represent assays occurring over a relatively short time frame, in part because the current set-up does not provide larvae with a food source. Ensuring adequate nutrition would be necessary to study changes in activity over extended periods of time or to measure circadian rhythm in the longer term. The 4% agar plugs may restrict gas exchange between the chamber and the external environment, which could result in the larvae experiencing hypoxic conditions. However this does not appear to affect activity within a 20 min assay period, because when average moves per minute of larvae during each minute of the period were analyzed, larvae did not appear to show any change in activity over the recording period (Figure 4).

Because the device records position continuously it represents an improvement in capturing more motion compared to the non-automated methods cited, however some motion does escape detection. Very small movements of animals might not trigger a response from this device, and larvae can move in a circumferential fashion within one infrared beam without breaking neighboring beams, resulting in inaccurately low readings. However, since this type of error would be expected to occur in all treatment groups, it is unlikely to cause misleading results. Although third instar larvae are the primary focus of this analysis, the device is capable of measuring the motion of the much smaller first and second instar larvae as well (Figure 3). As expected, the number of moves recorded per minute in the younger animals is lower than that of the larger third instar animals.

Although the full range of uses for this device has yet to be demonstrated, there are a variety of other adaptations that could diversify the uses of this apparatus for studies involving larvae. For instance, the device allows a ‘dwell’ measurement, which represents time spent in a determined region of the tube. This may provide valuable information when employed in various Drosophila larval taxis assays. By placing the apparatus on its side so that the tubes are oriented vertically instead of horizontally, one could measure larval geotaxis. To measure phototaxis, a light gradient could be established in the tubes, testing whether larvae have a preference for light or its absence. To study chemotaxis, a test chemical could be placed on one of the agar plugs and the position of the larvae might then reveal determine their preference for or avoidance of the chemical.

The monitor system allows the analysis of various motion parameters, summarized in Table 1. By selecting all parameters during the pre-assay setup (see step 3.6), the experimenter can choose which parameter to analyze after the assay. However, if any setting is not selected, that data will not be available for post hoc analysis. It should be noted that after each selected period of time, data are frozen at current counts and saved to the host computer. Data collection then resets to zero after this period and begins again, providing a series of time interval data points. One must manually quit to end data recording.

Future studies involving this method will focus on the use of the dwell parameter and its various applications. Also it may be possible to develop a protocol that would allow for studies to occur over a longer period of time, such as circadian studies, by providing food and exchanging agar plugs for a more gas-permeable material¹⁴. Moisture levels would need to be controlled as well, as dry conditions inhibit locomotion¹⁵. Currently, this protocol provides an accurate, simple, cost-effective method to evaluate basic parameters of larval activity under a variety of experimental conditions.

Materials

Drosophila Activity Monitor, Multibeam, 16 tubes, including wires	TriKinetics Inc.	MB5
Power Supply Interface for Activity Monitor	TriKinetics Inc.	PSIU24
Glass 80 x 5 mm tubes for Activity Monitor (100)	TriKinetics Inc.	PGT 5×80
DAMsystemMB1v6x Data Acquisitions Software for Macintonsh OSX (Intel)	www.trikinetics.com		free download
DAMFileScan 108x software for Macintosh	www.trikinetics.com		free download
USB software (PSIUdrivers.zip).	www.trikinetics.com		free download
DAMSystem Notes 308	www.trikinetics.com		free download
Zeiss Stemi 2000C- Stereo Microscope	Spectra Services	SP-STEMI2000C-BS
Carbon Dioxide	Maine Oxy		anaesthesia
Fly Pad	Genesee	59-114	surface for sorting anaesthetized flies
Small paint brush	Winsor & Newton	#2 ROUND	or similar, used for sorting anaesthetized flies
Silk Screen Printing Mesh (160)	msj-gallery.com	SM160W63-3YD	pore sized used in this protocol was ~ 0.1 mm
Tegosept	Genesee	20-258	preservative
Ethanol (190proof)	Pharmco	111000190	used to dissolve Tegosept
6 oz Square Bottom Bottle (PP)	Genesee	32-130
"Flugs" for Plastic Fly bottles	Genesee	49-100
Drosophila Vials, Wide (PS)	Genesee	32-117
Flugs for wide plastic vials	Genesee	49-101
Yellow Degerminated Corn Meal	Gold Medal
Drosophila agar	LabScientific	FLY 8020
Baker's Yeast – Red Star	King Arthur Flour	1270
Granulated Sugar – Extra Fine	Domino