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Rapid Escape Response Assay

AIM: To quantify the escape response of earthworms to a gustatory repellant (200mM NaCl).

INTRODUCTION: 200mM NaCl has been previously shown to be a strong gustatory repellant
in earthworm Eudrilus eugeniae (standardized by Amit. K. (2006), Ashwini. M. (2008), and
Viraj. T.(2009)).
Locomotion in earthworm is brought about by coordinated reciprocal contractions of
longitudinal and circular muscle bands which lie in the body wall. In the escape response assay
locomotion of the animal along the glass wall, away from the gustatory repellant is quantified.
The hyposthesis is that an animal with motor coordination dysfunction will have difficulty in
doing the same task.

MATERIALS:
Organism: Preclitellar worms of length 5 10cm in individual cup cultures,

Reagents: 200mM NaCl solution, distilled water.

Apparatus and miscellaneous: 30 test tubes (15ml), 10 ml pipette, test tube rack, Timer, glass
bowl with tap water, paint brush, filter paper.

METHOD:
1. Take a clean dry glass test tube. Put 1ml of solution (distilled water or 200mM NaCl) in it,
taking care that it doesnt touch the walls of the test tube.
2. With the help of a brush take out one worm from the cup culture gently and give it a brief wash
in the bowl with tap water to remove any adhering castings.
3. Pick up the worm with the same brush, roll it up and submerge it in the solution. Take care that
the worms do not touch glass wall and it is directly put into the solution.
4. Immediately start the stop watch; the time taken for the worm to climb completely out of the
solution is recorded for a maximum of 2 min.
5. Steps 1 4 are repeated for another 29 readings; a new tube is used for every reading. A
minimum of 30 readings are taken using 10 worms. Please note that the trails for one worm are
not given consecutively. The 10 worms are used in a loop to give each worm equal time to rest.
6. Escape Response Index, the percentage of worms that climbed within two minutes, is
calculated.

Rapid Escape Response =


Number of trails in which the animal climbs out completely in 2 min X 100
Total number of trails

Observed RER index for normal worms


a) With D/W (n=20): 10.68 +/- 8.96%
b) 200mM NaCl (n=37): 84.62 +/- 8.23%

Costing: 30ml of 200mM NaCl solution requires 3.39g of NaCl that costs just Rs.0.664 which
is the cost of running one assay with 30 readings.

Developed by Ramya. R., Viraj. T., Aparna. L., Malti. G., and Arunan M C

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