Cardiovascular Performance Measurement in Water Fleas by Utilizing High-Speed Videography and ImageJ Software and Its Application for Pesticide Toxicity Assessment

SIMPLE SUMMARY: With the advantages of easy culture, body transparency, and high sensitivity to chemical pollution, water fleas have been recognized as a good model for ecotoxicity studies. In this paper, we established ImageJ-based methods to measure cardiovascular performance by evaluating the heart rate and blood flow velocity in three water fleas for the first time. Among the three water fleas, Daphnia magna was identified as having the most robust heartbeat and blood flow rate and is therefore suitable for ecotoxicity assessment. Many important parameters like heart rate, blood flow rate, stroke volume, ejection fraction, fractional shortening, cardiac output heartbeat regularity can be extracted from videotaping and mathematical calculation. In utilizing those physiological parameters, the potential impacts of ambient water temperature and pesticide pollution on water fleas can be precisely measured. ABSTRACT: Water fleas are a good model for ecotoxicity studies, and were proposed for this purpose by the United States Environmental Protection Agency, due to their easy culture, body transparency, and high sensitivity to chemical pollution. Cardiovascular function parameters are usually used as an indicator of toxicity evaluation. However, due to the nature of the heart and blood flow, and the speed of the heartbeat, it is difficult to perform precise heartbeat and blood flow measurements with a low level of bias. In addition, the other cardiovascular parameters, including stroke volume, cardiac output, fractional shortening, and ejection fraction, have seldom been carefully addressed in previous studies. In this paper, high-speed videography and ImageJ-based methods were adopted to analyze cardiovascular function in water fleas. The heartbeat and blood flow for three water flea species, Daphnia magna, Daphnia silimis, and Moina sp., were captured by high-speed videography and analyzed using open-source ImageJ software. We found the heartbeat is species-dependent but not size-dependent in water fleas. Among the three water fleas tested, D. magna was identified as having the most robust heartbeat and blood flow rate, and is therefore suitable for the ecotoxicity test. Moreover, by calculating the diameter of the heart, we succeeded in measuring other cardiovascular parameters. D. magna were challenged with temperature changes and a pesticide (imidacloprid) to analyze variations in its cardiovascular function. We found that the heartbeat of D. magna was temperature-dependent, since the heartbeat was increasing with temperature. A similar result was shown in the cardiac output parameter. We also observed that the heartbeat, cardiac output, and heartbeat regularity are significantly reduced when exposed to imidacloprid at a low dose of 1 ppb (parts per billion). The blood flow rate, stroke volume, ejection fraction, and fractional shortening, on the contrary, did not display significant changes. In conclusion, in this study, we report a simple, highly accurate, and cost-effective method to perform physiological and toxicological assessments in water fleas.