1	The Development of an Assay to Accurately Measure the Effect of Acetylcholine, Caffeine, and Nicotine on the Heartbeat Rate of the Primitive Crustacean Triops longicaudatus in the Larval Stage Scott Harden
2	Triops longicaudatus Freshwater crustacean that inhabits temporary water pools formed by rainfall Referred to as “living fossils” Unique life cycle Eggs Larval stage Adult stage Diapause
3	Triops heartbeat Crustacean Heart Generally possess a neurogenic heartbeat A cardiac ganglion serves as the pacemaker Nerve fibers accelerate or inhibit heartbeat Triops Heart Recently proven to lack cardiac ganglion Heartbeat rate controlled myogenically Myocardium serves as pacemaker
4	Triops heartbeat research Triops heartbeat can be difficult to study Heartbeat rate very sensitive to O₂ level Small size of larval stage makes study difficult
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7	Triops heartbeat research Triops heartbeat can be difficult to study Heartbeat rate very sensitive to O₂ level Small size of larval stage makes study difficult Triops larvae are extremely fragile Heartbeat rate is modified by pressure Beat rates can exceed 600 BPM Heart usually removed from body of adults Research Goal Develop a method to accurately measure the heartbeat rate of live Triops in the larval stage and test this method by analyzing the effects of caffeine, acetylcholine, and nicotine on the heartbeat rate
8	Flow-through stage schematic
9	PIC
10	Flow-through system schematic
11	PIC2
12	Video Analysis of Triops Heartbeat A stream of video is recorded that documents heartbeat activity before and while an experimental solution is administered (~4 min) Video is enhanced to accentuate heart Two layers of high contrast filters Converted to grayscale and inverted Frame rate decreased to 15 FPS Sliced into 10 second clips Frames excised to eliminate partial beats Number of full beats counted Heartbeat rate calculated (BPM) Accurate to 2 BPM for a 5 second video recording
13	VID 1
14	VID 2
15	VID 3
16	Statistical Analysis Heartbeat Rates Heartbeat rates in control solution are plotted Linear regression equation is found to predict future heartbeat rates
17	Statistical Analysis Heartbeat Rates Heartbeat rates in control solution are plotted Linear regression equation is found to predict future heartbeat rates
18	Statistical Analysis Heartbeat Rates Heartbeat rates in control solution are plotted Linear regression equation is found to predict future heartbeat rates Experimental solution deviance from predictions measured by chi-square analysis
19	Experimental Results The method described can Accurately measure larval Triops heartbeat rates This method allows for the effects of various solutions on the heartbeat rate of a larval Triops to be and easily determined without harm to the organism being studied
20	Control Solution
21	10 ppm Caffeine Solution
22	10 ppm Acetylcholine Solution
23	10 ppm Nicotine Solution
24	VID 4
25	Future Research Discover the effects of different chemical solutions (or alternate concentration levels) on the heartbeat rate of larval Triops Determine the effects of various concentrations of nicotine on the paralysis of larval Triops Measure the effects of high levels of acetylcholine to determine if small amounts of neurogenicity exist in the larval Triops heart
26	Acknowledgements Dr. Wayne Wofford Dr. Andrew Madison Dr. Michael McMahan Angelina Ramírez Kent Willis Union University Biology Department
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