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1. DONE WITH EVERYTHING & EVERYONE Mood:. . . Abstract # 0000 The Effects of EDTA/Tetracycline on Wolbachia Pipientis Michael Sturdivant and Edwin Chavez E.E.Waddell High School Observations and Data Abstract Experimental Methods Since its discovery, Wolbachia has garnered a spike in interest among the scientific community due to its gender bending characteristics and its growing associations with human filarial diseases, such as Lymphatic Filariasis (figure B), and Onchocerciasis (figure C) These gram- endosymbionts reside within reproductive systems of invertebrates and filarial nematodes. As these associations are beneficial to Wolbachia their hosts experience negative effects like virgin births, skewed gender ratios, cytoplasmic incompatibility (figure A), and lymphatic inflammation within secondary hosts like humans. While strides have been made to cure these uncultivable organisms insitu with Tetracycline, attempts for total eradication are necessary to make strides in reduction of human inflammatory responses. To assess the synergistic effects of EDTA and Tetracycline on Wolbachia, four culturing vials were prepared with different treatment combinations( 4% Sucrose solution, .005% EDTA/4% Sucrose solution, 0.031mg/ml Tetracycline/4% Sucrose solution, and 4% Sucrose/.005% EDTA/0.031mg/ml Tetracycline solution). Each vial was populated with descendents of Wolbachia infected Nasonia wasps (5 females/ 2 males) and sarcophaga hosts. After mating, parent wasps were removed, & assessed for Wolbachia via DNA extraction, amplification of Wolbachia 16s rRNA gene and gel electrophoresis. Upon hatching, all offspring gender was assessed and used with similar treatments to set up succeeding generations. Our data showed that EDTA/Sucrose not EDTA/Tetracycline eradicated and reversed the gender skewing effect Wolbachia. This treatment increased male presence by 30% and reinstated gender balance. In conclusion EDTA is an effective agent to minimize the effects of Wolbachia in its primary and secondary hosts. 1.)The treatment vials were set up as follows: 4% Sucrose solution, 5 females, 2 males (control). .005% EDTA/4% Sucrose solution, 5 females, 2 males. Figure 3 shows the succeeding generations of wasps with a 0.005% EDTA/0.0031mg/100ml Tetracycline/4% Sucrose treatment. Figure 1 shows the succeeding generations of wasps with a 4% Sucrose treatment. Figure 2 shows the incidence of Wolbachia infections in treated parents of offspring.( All Treatments). Figure A 4% Sucrose/.005% EDTA/0.031mg/ml Tetracycline solution, 5 females, 2 males 0.031mg/ml Tetracycline/4% Sucrose solution, 5 females, 2 males • Post copulation parents were removed. 2.)Using the Qiagen Dneasy Blood and Tissue Kit we extracted DNA from the parents. 3.) The 16S RNA region of W.Pipientis was amplified. Figure 5 shows the succeeding generations of wasps with a 0.0031mg/100ml Tetracycline/4% Sucrose treatment. Figure 4 shows the succeeding generations of wasps with a 0.005% EDTA/4% Sucrose treatment. Results and Conclusions Our data showed that EDTA/Tetracycline/Sucrose was unable to eradicate and reverse the gender skewing effect of Wolbachia Pipientis. In fact, this antibiotic/chelating combination proved to be metabolically debilitating, which for example led to early mortality in adults treated under these conditions (the wasps died before copulation). (Figure 3) .Of the only viable generation, one parent wasp was infected with Wolbachia endosymbiont. Thus proving that this treatment combination was too metabolically demanding on the Nasonia hosts. ( Figure 3). Though the predicted combination and its effects were not manifested in the study, one constituent, EDTA proved effective at not only eradicating the parasite (See figure 2) but reversing male/female skewing ratio as well. (Figure 4). Graphical analysis shows that when compared to the control treatment, EDTA/Sucrose had a 30% difference and increase in the males presence in the final generation compared to this control, which supersedes our hypothesis of at least a 10-20% increase. (Figures 1&2). Thus, demonstrating EDTA’s ability to be an effective agent on Wolbachia pipientis. Figure D 4.) PCR products were then processed on a 0.8% Ethidium Bromide gel References Acknowledgements 1.) Borndenstein, Seth R., Julieanne J. Uy, and John H. Werren. "Host Genotype Determines Cytoplasmic Incompatibility Type in the Haplodiploid Genus Nasonia." NCBI The University of Rochester, 15 Jan. 2003. Web. 6 Feb. 2010. 2.) Hoerauf A, Nissen-Pahle K, Schmetz C, Henkle-Duhrsen K, Blaxter ML, et al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. J Clin Invest 103: 11–18. 3.) Foster J, Ganatra M, Kamal I, Ware J, Makarova K, et al. (2005) The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biol 3: e121. doi:10.1371/journal.pbio.0030121. 4.) Brownlie JC, O'Neill SL (2005) Wolbachia genomes: insights into an intracellular lifestyle. Current Biology 15: R507–509. 5.) Lambert, R. W., Hanlon, G. W., & Denyer, S. P. (2004). The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa. The Science of . Applied Microbiology, 245-253. 6.) Nasonia Genetics- A Jewel of a Wasp (n.d.). Retrieved November 4, 2009, from http://www.biologycorner.com/worksheets/nasonia.html 7.) Pike, Nathan, and Rachel Kingcombe. "Antibiotic treatment leads to the elimination of Wolbachia endosymbionts and sterility in diplodiploid collembolan Folsomia candida." BMC Biology N.p., 24 Aug. 2009. Web. 24 Nov. 2009. 8.) Knight, Jonathan. "Meet the Herod bug." Macmillan Magazines Ltd 5 July 2001: 12-14. Print. 9.) Warren, John H. "Biology of Wolbachia." N.p., 1997. Web. 24 Nov. 2009. 10.) Bordenstein, Seth. The Bordenstein Lab Vanderbilt, 2008. Web. 24 Nov. 2009. <http://bordensteinlab.vanderbilt.edu/>. 11.) EDTA. (2002). Retrieved February 6, 2010, from http://medical-dictionary.thefreedictionary.com/Calcium+disodium+ethylene+diamine+tetra-acetate • Tamica Stubbs, E.E.Waddell High School Charlotte,NC • Dr.Seth and Sarah Bordenstein, Vanderbilt University Nashville,TN • Figure A courtesy of http://dobsonserv.ca.uky.edu/DobsonSite/CI1.html • Figure B courtesy of lepre.org/uk • Figure C courtesy of http://www.csiro.au/files/mediaRelease/mr2000/Riverblind.htm • Figure D courtesy of http://www.molecularstation.com/molecular-biology-images/509-pcr-pictures/73-pcr-polymerase-chain-reaction.html?size=big • Additional photos by Michael Sturdivant and Edwin Chavez 5.)Compare and contrast male to female ratio data and presence of Wolbachia over the successive generations. Repeat steps for succeeding generations. 6.) Chi Square analysis was performed. Figure B Figure C Future Implications Hypothesis • Wolbachia has been linked to human diseases such as Lymphatic Filarisis (Elephantiasis) and Onchocerciasis (River Blindness). • Studies on EDTA as an fighting agent on such human diseases Wolbachia has been known to cause. • EDTA treatments to prevent mosquito borne diseases. • Wolbachia’s effect on the ecosystem. If EDTA/Tetracycline treatment is used to treat Wolbachia infections in Nasonia wasps, then we will see a 10-20% increase of males in the final generation.