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I was dumbstruck by the image of the green neurons in the purple background as one of my lab mates presented his weekly data. This was my first week at the Gordon Lab and nothing had elicited more excitement than the confocal image of the fly brain! (September, 2018)
Behavioral Assays
Fly Two-Choice Assay: In the fly two-choice assay, we would provide the flies with choices of two different tastants, and qualitatively score their preference based on the color of their abdomen after the feeding period (2 hours). We mixed the tastant of choice with 1% agar and food dye. If the fly consumed the tastant mixed with red dye, its abdomen would turn red and if it consumed the tastant mixed with blue dye, its abdomen would turn blue, and if it consumed both, we would see a shade of purple. Using this assay we could qualitatively decipher their taste preferences.
NOTE: To remove color bias we would place the flies in a dark chamber with controlled temperature and humidity during the feeding period of 2 hours. We also alternated the color of the tastants in the experiments.
We used 24 hours starved flies for this assay (except for UAS-Kir2.1 crossed flies).
Proboscis Extension Response (PER): This is an appetitive assay where the flies would extend their proboscis when stimulated with an appetitive tastant. The flies would be stuck inside a pipette tip with only their heads sticking out. This preparation would be stuck on a slide with double-sided tape. The flies were allowed a recovery time of 30 minutes inside a humid box before the experiment was conducted and the flies were fully satiated.
During the experiment, we touched the fly's proboscis with the tastant and noted its response. If the fly extended its proboscis, that meant the tastant was appetitive. For aversive or neutral tastants, the fly would not show any response.
Illustration of fly two-choice assay
Illustration of mosquito two-choice engorgement assay (left)
Mosquito Two-Choice Engorgement Assay: We tried to adapt the fly two-choice assay to evaluate mosquito preference. However, due to the hematophagous nature of the mosquitoes and prominent sexual dimorphism, we had to change the design and craft a novel assay. (Dr. Ben Matthews generously allowed me to utilize his brand new lab space to conduct these experiments and provided constant feedback and advice.)
We used glytube feeders with stretched parafilm to mimic human skin and used minimal meal instead of blood for the ease of scoring the mosquitoes on the basis of their colored abdomen. We lined these feeders with a mesh drenched in the tastant of choice.
Below is a video of the assay setup and the scoring process.
We also designed the two-choice feeding assay mimicking nectar feeding behavior to evaluate the preference towards lactic acid in male and virgin female mosquitoes. For this assay we used cotton wick soaked in 10% sucrose solution layered with a mesh drenched in lactic acid.
Illustration of mosquito two-choice feeding assay
Mosquito Engorgement Assay.mp4Video protocol of mosquito two-choice engorgement assay
Fly and Mosquito Husbandry
Fly Husbandry: Fly pushing; familiarity with genetic markers; regular flipping and setting up of crosses.
Mosquito Husbandry: Familiarity with fluorescent markers; regular turning over and setting up of crosses.
Life of an experimental mosquito (Illustrated using Adobe Illustrator)
Fly brain with GCAMP6 labeled 'sweet' gustatory neurons (Gr64f)
Microscopy
I have conducted whole-brain dissections and microsurgeries for live calcium imaging. During calcium imaging, we would stimulate the fly proboscis with a panel of tastants and image the subeosophageal ganglion zone (SEZ) of the fly brain where the peripheral taste neurons converge.
Programming Skills
My undergraduate training in computer science has made me proficient in C-language. I currently have intermediate-level expertise in Python Programming. I found some of the resources from Dr. Russ Polrack's blog super helpful when I started learning Python programming through most of COVID -19.
Others
Adobe Photoshop, Adobe Illustrator, Image J
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