MICROFLUIDIC CAPILLARY FEEDER WITH INTEGRATED ACTIVITY MONITORING CHAMBER FOR DROSOPHILA STUDIES Deepak Choudhury1, RapeechaiNavawongse2, MarlenaRaczkowska2, Zhiping Wang1, Adam Claridge-Chang2,3,4 [email protected]
SIMTech A*STAR, Singapore 2 IMCB A*STAR, Singapore 3 Duke-NUS Graduate Medical School, Singapore 4 Department of Physiology, National University of Singapore, Singapore ABSTRACT We have developed a novel microfluidics chip based platform to study flies feeding behaviour. The microfluidic channel is designed to accurately feed a miniscule meal size to the flies. One-day feeding portion/supply can be stored inside the chip without refill. Our camera, panel holder and chip holders make up the Espresso hardware system and are designed for high-throughput assay with higher fidelity where up to 30 flies can be monitored simultaneously with a single camera. KEYWORDS: drosophila, fly-on-a-Chip, activity monitoring, fly feeding, fly behaviour INTRODUCTION Feeding is the one of the most common behaviour among living beings from mammals to single cell organisms. The foraging strategies and choices of food have been studied intensively in various species [1-3]. The molecular and behavioural mechanism driving the feeding behaviors however is still unclear. Drosophila melanogaster has been widely used as model for biological studies for well over a century .We have developed a new microfluidics chip based platform [Figure 1A-B] to study flies feeding behaviour. The microfluidic channel is designed to feed a miniscule meal size of 0.08 ul per meal on average. One-day feeding portion/supply can be stored inside the chip without refill. Two channels allowing preference studies have been integrated into one behaviour chamber, where the fly activity is continuously monitored. Knowing the fluid level during the period of experiment allows determining the precise feeding time, feeding bout and feeding amount for a single fly. The platform allows to probe into several basic Drosophila behaviors, namely, walking, sleeping, mating, foraging and feeding simultaneously. EXPERIMENTAL Each chip was fabricated using transparent thermoplastic cast acrylic material (PMMA- Poly (methyl methacrylate). Computer numerical control (CNC) milling machine was utilized to fabricate various layers of the chip. Thermal fusion bonding was employed to bond the various layers of the chip. After thawing the assembly, tapping was done for the two holes at the corners of the chip. This assembly was tested for any leaks by dispensing coloured food dye through all the channels. The chip holders, panel holder and camera make up the necessary assay system [Figure 2A-B]. Entire system was accommodated inside the incubator, which control light, temperature, and humidity. All flies used in our experiments were out-crossed with CS strain for 5 generation. Four Gal4 drivers: TH-Gal4, Tdc2Gal4, Trh-Gal4 and CS-Gal4 flies- were crossed with UAS-Kir2.1; tub-Gal80ts to produce F1 progenies used on behavioral experiments. F1 progenies were maintained at 22°C, 60-70% relative humidity, under 12:12 hour light and dark cycles for 4 to 7 days before the experiment day. Kir2.1 was expressed by incubating flies at 31° C for 24 hours. The red fluid with 5% sucrose was loaded in to the fluid channel before loading the flies. The experiment was recorded and analyzed off-line. RESULTS AND DISCUSSION The system is suitable for high-throughput assays; where up to 30 flies can be monitored simultaneously. An in-house GUI MATLAB program [Figure 3] has been developed to analyze the height of fluid in the channel and location of Drosophila. The usefulness of the system was
19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA
demonstrated by the neuromodulatory control of feeding study [Figure 4]. Candidate neurotransmitters known to influence feeding were tested for their role in Drosophila feeding behaviour. We used conditional expression of Kir2.1 to silence the specific set of neurons.When flies were raised at permissive temperature (no inc=no incubation), Gal80ts was expressed, inhibiting expression of Kir2.1. When flies were shifted to restrictive temperature for 24h (inc= incubation), Gal80ts was inactive, allowing expression of Kir2.1, resulting in the neurotransmitter deficiency. We found that dopamine deficient flies (TH inc) showed similar to control fluid consumption upon 24-hr starvation under both conditions. However, octopamine deficient flies (Tdc2 inc) showed reduced feeding and flies that lack serotonin (Trh inc) showed increased feeding. B
Figure 1: Espresso Chip: (A) Chip with all its layers (solid model and wireframe model).The chip has four layers and bonded together under a custom jig (B) Detailed description of layer 3 of the chip. Each fly chamber has two microfluidic channel feeders. The bottom humidity chamber (water reservoir) keeps higher humidity within the chamber and reduces fluid evaporation. A
Figure 2: Espresso Hardware System :(A) Isometric view (B) Back view CONCLUSION Almost 1500 flies have been tested on the multiplex system. Our system can provide researchers with many useful metrics within one experiment, saving time and cost. Since our assay can control the food given to the flies, one can control dose, concentration and time of drug delivery to Drosophila through its gastrointestinal tract. With all the benefits mentioned, this device would be useful in many areas including drug screening, feeding disorders and sleep studies.
Figure 3: Our in-house GUI MATLAB program used to analyze a video file. The program calculated liquid level and location of flies on each channel. The region of interest (ROI) can be selected as shown in green and yellow square borders on the chip image.
Figure4: Neuromodulatory control of feeding. Candidate neuromodulators (Octopamine –tdc2, dopamine -TH, and Serotonin- Trh) were tested for their role in feeding behaviour upon conditional expression of Kir2.1 (incubation=inc), resulting in the neurotransmitter deficiency. All flies were starved for 24hr and tested with 5% sucrose fluid food. n=~30. ACKNOWLEDGEMENTS The project has been kindly supported by the A*STAR Joint Council (JCO) Grant, Singapore. We would like to thank Emma Morris and Ng Tzer Liang (SIMTech) for their help in the design of the Espresso hardware system. REFERENCES Bixter MT1, Luhmann CC. (2013) “Adaptive intertemporal preferences in foraging-style environments.” Front Neurosci. Jun 17; 7:93. Hölzl M1, Krištofík J, Darolová A, Hoi H. (2011) “Food preferences and mound-building behaviour of the mound-building mice Musspicilegus.” Naturwissenschaften. Oct; 98(10):863-70. Lima JT, Costa-Leonardo AM. (2014) “Foraging in subterranean termites (Isoptera: Rhinotermitidae): how do Heterotermes tenuis and Coptotermesgestroi behave when they locate equivalent food resources?” Bull Entomol Res. Aug; 104(4):525-33. Dussutour A, Simpson SJ. (2009) “Communal nutrition in ants.” Curr Biol. May 12; 19(9):740-4. Rubin GM, Lewis EB. (2000) “A brief history of Drosophila's contributions to genome research.” Science. CONTACT: [email protected]
The capillary chip and the associated assay system have patents pending. 727