1. Using Molecular Analysis to Examine the Phylogeny of Symbiotic Water Mites in Two Subgenera: Unionicoloides and Parasitax Dan Deatherage Kevin Myers September 3,2004

2. Background Symbiotic water mites Mussels provide hosts to mites Size requires molecular techniques Molecular phylogeny Vs. Classical Phylogeny Cytocrome Oxidase I (COI) gene Vs. Internal Transcriber Sequence (ITS)

3. Why These Mites? Readily found and collected Generally many individuals in each population Mites similar to these have been heavily studied The COI sequences for these mites are not known This field of study is open to new research without fear of being “scooped” The dissemination of species was begun by Dr. Edwards

4. Unionicola sp. All species of Unionicola are morphologically very similar Utterbackita imbecillis Anadonta suborbiculata

5. Proposed Research Project begun through the UExplore Undergraduate Research Program summer 2004. Mites chosen because their close classical phylogeny classification. Results during the summer have shown that the methods we will use are effective.

6. Species of Mites to Study Include: From the subgenus Parisitax: –Unionicola formosa from Pyganadon cataracta –U. formosa from P. grandis From the subgenus Unionicoloides: –Unionicola kavanaughi –U. gailae –U. hoesei –U. lasallei –U. amandita

7. Stored Mites & Predetermined Primers For Each Mite

8. Primer Design with NetPrimer

11. Stored Mites & Predetermined Primers PCR Extracted DNA For Each Mite

12. Ladder |–––––1-4––––––| |–––––5-8––––––| |–-––––9-12–––-––| Gel electrophoresis of U. foili from U. imbecillis (1-8) and U. formosa from A. suborbiculata (7-12). There are bright bands for each mite, supporting the near correct sequence of our primers.

13. Stored Mites & Predetermined Primers PCR Extracted DNA Sequence Purified PCR Product For Each Mite

14. CodonCode Aligner

15. Stored Mites & Predetermined Primers PCR Extracted DNA Sequence Purified PCR Product Create a Molecular Phylogeneic Tree For Each Mite

16. ClustalW

17. Molecular Phylogenic Tree

18. Timeline Sept 15Preliminary extraction underway. All materials gathered. Sept 30All DNA extracted from initial mites and 2 species sent for sequencing. Oct 152 more species sequenced, initial 2 species sequence analysis began. Oct 30Final 2 sequences returned, analysis continuing. Nov 15All sequence analysis complete, manuscript begun. Nov 30All lab work done, and manuscript in final stages. Dec 15Manuscript submitted for class and for publication.

19. Budget ItemCost DNA Sequencing $300 (16 sequences) Qiagen DNeasy Tissue Kit $156 Promega PCR Master Mix $67 (100 reactions) Total: $523

20. Grade Agreement Level of Progress Achieved Points Received DNA successfully extracted 3.0 PCR product successfully amplified 4.0 Sequence data successfully returned 1.5 Sequence data successfully analyzed 4.0 Total Points for Each Mite Species 12.5 Remaining Happy Throughout the Semester Priceless

21. References Boore, J. L. 1999. Animal Mitochondrial Genomes. Nucleic Acids Research, 27(8):1767-1780. Edwards, D. D. and Ernsting, B. R. 2004. UExplore Undergraduate Research Proposal. Myers, Kevin, Eyler, Andrea, and Rasure, Mark. 2004. Unpublished Data. Navajas, M. and P. Boursot. 2003. Nuclear ribosomal DNA monoplyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life history and molecular drive. Proceedings of the Royal Society of London B Biological Sciences, 270 Suppl 1:S124-127.

22. Questions? Ideas?