قياس اليرقات آخر في<em> ذبابة الفاكهة</em> مراقبة النشاط
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 time6-7. 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
Representative Results
Discussion
ويتأثر النشاط من يرقات ذبابة الفاكهة من قبل مجموعة متنوعة من العوامل بما في ذلك النمط الجيني 8، سن 13 ودرجة الحرارة المحيطة 2. على الرغم من أن وسائل videographic قوية قادرة على تحليل مفصل للغاية تم تطويرها من قبل أولئك الذين يدرسون الحركة 5، هذا ال…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIH P20GM103643 to I. Meng.
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 |
Riferimenti
- Tracey, W. D., Wilson, R. I., Laurent, G., Benzer, S. painless, a Drosophila.gene essential for nociception. Cell. 113, 261-273 (2003).
- Ainsley, J. A., Kim, M. J., Wegman, L. J., Pettus, J. M., Johnson, W. A. Sensory mechanisms controlling the timing of larval developmental and behavioral transitions require the Drosophila.DEG/ENaC subunit. Pickpocket1. Dev. Biol. 322 (1), 46-55 (2008).
- Pandey, U. B., Nichols, C. D. Human disease models in Drosophila. melanogaster.and the role of the fly in therapeutic drug discovery. Pharmacol. Rev. 63 (2), 411-436 (2011).
- Cattaert, D., Birman, S. Blockade of the central generator of locomotor rhythm by noncompetitive NMDA receptor antagonists in Drosophila.larvae. J. Neurobiol. 48 (1), 58-73 (2001).
- Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. Dynamic analysis of larval locomotion in Drosophila. hordotonal organ mutants. PNAS. 100 (26), 16053-16058 (2003).
- Nichols, C. D., Bechnel, J., Pandey, U. B. Methods to assay Drosophila behavior. J. Vis. Exp. (61), (2012).
- Godoy-Herrera, R. The development and genetics of digging behavior in Drosophila. arvae. Heredity. 56, 33-41 (1986).
- Sinadinos, C., Cowan, C. M., Wyttenbach, A., Mudher, A. Increased throughput assays of locomotor dysfunction in Drosophila. larvae. Journal of Neuroscience Methods. 203 (2), 325-334 (2012).
- Homyk, T., Sheppard, D. E. Behavioral mutants of Drosophila melanogaster. I. Isolation and mapping of mutations which decrease flight ability. Genetica. 87 (1), 95-104 (1977).
- Chattopadhyay, A., Gilstrap, A. V., Galko, M. J. Local and global methods of assessing thermal nociception in Drosophila. larvae. J. Vis. Exp. (63), e3837 (2012).
- Model Organisms I: yeast, Drosophila and C. elegans. Science Education Database Available from: https://www.jove.com/science-education-database/3/essentials-of-biology-1-yeast-drosophila-and-c-elegans (2015)
- Woods, J. K., Kowalski, S., Rogina, B. Determination of the spontaneous locomotor activity in Drosophila melanogaster. J. Vis. Exp. (86), e51449 (2014).
- Troncoso, B., Godoy-Herrera, R., Waldo, M. The development of larval movement patterns in Drosophila). Heredity. 58 (1), 321-329 (1987).
- Helfrich, C., Engelmann, W. Circadian rhythm of the locomotor activity in Drosophila. melanogaster.and its mutants ‘sine oculis’ and ‘small optic lobes. Physiological Entomology. 8 (3), 257-272 (1983).
- Johnson, A. W., Carder, W. J. Drosophila, ociceptors mediate larval aversion to dry surface environments utilizing both the painless TRP channel and the DEG/ENaC subunit, PPK1. PLoS ONE. 7 (3), (2012).
