Background.

The purpose of the proposed robotic system is to decrease a labor intensive research process that will increase data throughput while at the same time freeing labor time to be spent in data analysis. At the Tufts New England Medical Center site, graduate student Kerry Garrity is conducting research on fruit flies in an attempt to make conclusions about human genetics. Her research is currently testing the effect of a certain serotonin gene, 5HT, on the overall food intake of fruit fly larvae. As so far, Kerry can test 50 different genetic lines each week, but the desired data range is up to 10,000 mutant lines obtained from Bloomington Drosophila Stock Center at Indiana University.

Fruit flies are being used because they contain positive attributes for the requirements of the research. When studying food intake, it is important to isolate and control variables that could disrupt the results, such as locomotor activity and feeding time. Drosophila larvae in the third instar stage, or three days after incubation, are ideal because they continually eat for the two days of that stage. Their transparent cuticle also allows for good visualization which allows for image analysis of the dyed intestinal track.

For that past year, Kerry has been defining her assay and taking preliminary data. Each genetic line is hatched until the larvae reach the third instar stage after which they are removed by hand and placed into a dyed food. The food components are a Drosophila Instant Food (Carolina Biological Supply Co.) base with 10% Brewer’s inactive yeast (ICN Biomedical) and 0.2% fast green dye. The larvae are tested between the hours of 8 and 10 am for data consistency because of various feeding patterns throughout the day. After eating the dyed food for 30 minutes at 25°C, 8 larvae are removed, rinsed, cleaned, inserted into a vial, and crushed into solution. Once two vials of 8 larvae are created to monitor for discrepancies, an optical density (OD) reading at the maximum emission wavelength of the dye (625nm) is taken on the supernate. A higher OD reading implies more food consumption for the tested genetic line.

Initial work and research were conducted in the summer of 2004 to find a testing system equivalent to the current OD readings. It was concluded that the food intake could be calculated by viewing the intestinal track through the transparent cuticle. Fluorescein acts as an ideal dye because it emits at a different wavelength than its excitation wavelength, therefore, allowing for the excitation light to be filtered leaving only the dye’s spectrum. Additional benefits include fluorescein’s low cost and non-toxic nature. A prototype system was constructed where the fluorescein fed larvae were excited by 400nm UV LEDs and then filtered by a 550nm low-pass filter before being analyzed by an ELMO linear 8-bit camera. Data collection was mirrored against Kerry’s and the 0.2% fast green dye was replaced with 0.2% fluorescein solution. Initial tests were conducted and carried into the fall semester for future exploration.