Studying Cocoa Polyphenols in Caenorhabditis elegans using Lifespan Assays Kristin Hausen, Dr. Kirsten Crossgrove Department of Biological Sciences, University of Wisconsin-Whitewater Introduction Caenorhabditis elegans was chosen for this research project as they are easy to maintain in the lab and have a relatively short lifespan. C. elegans lifespan assays are important to human biology because some of the mechanisms that control lifespan in C. elegans are conserved in humans (Tissenbaum, 2015). One of those mechanisms is a transcription factor named SKN-1,that extends lifespan in C. elegans and functions similarly to Nrf1,2 transcription factors in humans.(An and Blackwell, 2003). Many nutritional supplements have been shown to extend lifespan in C. elegans. I am interested in cocoa polyphenols. Polyphenols have been seen to aid in antioxidative repair mechanisms in cells as humans age (Pandey and Rizvi, 2009). Cocoa powder has also been shown to significantly extend lifespan in rats (Bisson, 2008). Two different concentrations of cocoa polyphenols (4% and 12%) extended lifespan in C. elegans (Martorell et al., 2011). I am using the CocoaVia Brand cocoa extract with 375mg of polyphenols per serving. I tested that lifespan extension can be achieved by using a cocoa powder media in fem-1 (wild-type phenotype but do not reproduce at 25ºC) and will also test the same conditions with skn-1 RNAi worms. Results: Cocoa Powder Increases Lifespan Figure 3: This graph is a survival curve of each of the three experimental conditions: control, 2g/L cocoa powder and 4g/L cocoa powder. All C. elegans in this experiment were fem-1 worms. Here we can see that the survival function for the control group (shown in blue) is substantially different than the 2g/L and 4g/L conditions. How dsRNAi Works and Why We Need to Use It. Cocoa Polyphenols Can Extend the Lifespan of C. elegans Figure 5: this figure depicts how dsRNAi can induce a mutant phenotype without C. elegans actually carrying the mutation. Source: Biomica Biotech Mechanism of RNAi http://www.biomics.com/en/AboutRNAi_1.html The reason that RNAi is used to induce a SKN-1 deficiency is because a mutation in SKN-1 itself is lethal in the embryonic stage of the life cycle. RNAi ensures that we have viable offspring while still having the effect of the mutation. In C. elegans we can easily induce RNAi by feeding it to them through E. coli transfected with the RNAi sequence for SKN-1. Conclusions Through lifespan assays, we were able to conclude that cocoa powder does in fact extend lifespan in C. elegans in the concentrations of 4g/L and 2g/L. We were also able to conclude that the extension seen in 4g/L and 2g/L concentrations did not significantly differ from each other. Hypotheses Cocoa powder in concentrations of 4g/L and 2g/L will extend lifespan in C. elegans. I expect to see a lifespan extension in the experimental conditions using cocoa powder over the control condition. Skn-1 is needed for lifespan extension to take place using cocoa powder. In Skn-1 RNAi mutants, I expect to see no extension in lifespan under the cocoa powder conditions. Future Directions Be able to digest the L4440 vector completely and insert the mutagenized skn-1 PCR product. Transform E.coli with the completed L4440/SKN-1 mut plasmid. Run another lifespan experiment with SKN-1 RNAi worms and control worms in both control conditions and cocoa powder conditions. Use SPSS to run a Life Table analysis to compare the lifespans SKN-1 RNAi worms to wild type worms in both control and cocoa powder conditions. Testing the Role of SKN-1 in Lifespan Extension Setting Up Aging Assays With Cocoa Powder Poured plates (three conditions: Control (NGM agar), 2g/L cocoa powder/NGM agar and 4g/L cocoa powder/NGM agar. Cocoa powder was diluted in 5mL water and sterile filtered after NGM was autoclaved) Age synchronized L1 larvae. Transferred ~20 worms to each NGM agar plate and allowed to develop to adulthood (15ºC). Once adulthood was reached, transferred worms to new plates with heat-killed OP50 (E. coli) and stored at 25ºC. Checked plates almost every day to count and transfer worms to new plates. Recorded how many worms were alive and how many worms had died. If a worm is poked and unresponsive it was scored as dead. Lane 1 Lane 2 Lane 3 Ladder Cut Vector Uncut Vector Works Cited Bisson, J.-F., Nejdi, A., Rozan, P., Hidalgo, S., Lalonde, R., & Messaoudi, M. (2008). Effects of long-term administration of a cocoa polyphenolic extract (Acticoa powder) on cognitive performances in aged rats. British Journal of Nutrition, 100, 94–101. Pandey, K. B., & Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity, 2(5), 270–278. Tissenbaum, H. A. (2014). Using C. elegans for aging research. Invertebrate Reproduction & Development, 59(1), 59–63. Figure 5: The reason for this gel was to ensure that we were able to successfully cut the vector using a restriction enzyme digest. We needed to cut the vector so that we could insert the skn-1 product created in figure 4. Lane 2 of this gel shows us the restriction enzyme digested L4440 vector while lane 3 shows us the undigested vector. Figure 4: This DNA Gel Electrophoresis shows a PCR product that was made for a RNAi construct we created to induce a skn-1 deficiency C. elegans as skn-1 mutants are lethal during the embryonic stage of development. Image Source: https://data.broadinstitute.org/bbbc/BBBC010/ Acknowledgements UW-Whitewater Undergraduate Research Program UW-Whitewater Biology Department Austin Chriske, Amanda Danno, and Johnna Dykstra for assistance in the lab Figure 1: Here we see a few C. elegans in which many of them are alive. You can tell by their more curved shape. Figure 2: Here we see many C. elegans in which all seem to be dead beginning to decay. Death is characterized by mostly straight bodies.