1. The Emerging Role of Molecular Markers in Coccidian Speciation K. B. Miska, D. Motriuk-Smith, and R. S. Seville The 83 rd Annual Meeting of the American Society of Parasitologists 40 th Coccidosis Conference and Workshop June 29 th, 2008 Arlington, TX

2. Partial ORF470 Reported to distinguish lineages that are oocyst residuum (OR) possessing or lacking (Zhao and Duszynski 2001) Reported to distinguish lineages that are oocyst residuum (OR) possessing or lacking (Zhao and Duszynski 2001) Generated new partial ORF470 sequences (sciurid rodent hosts) Generated new partial ORF470 sequences (sciurid rodent hosts) only E. callospermophili possesses ORonly E. callospermophili possesses OR

3. OR - murid hosts OR + sciurid and murid host OR - sciurid host unpublished ? A B NJ tree

4. ITS-1 and ITS-2 Reported to distinguish morphologically similar Eimeria species (Hnida and Duszynski 1999) in murid rodents Reported to distinguish morphologically similar Eimeria species (Hnida and Duszynski 1999) in murid rodents Generated new sequences to distinguish differences between morphologically similar species of Eimeria (sciurid rodent hosts) and/or diverse geographic locations Generated new sequences to distinguish differences between morphologically similar species of Eimeria (sciurid rodent hosts) and/or diverse geographic locations E. vilasi and E. pseudospermophili (3 hosts; WY)E. vilasi and E. pseudospermophili (3 hosts; WY) E. lateralis (4 hosts; WY, AK, Siberia)E. lateralis (4 hosts; WY, AK, Siberia) E. callospermophili (3 hosts; WY, CO)E. callospermophili (3 hosts; WY, CO)

5. E.vilasi and E.pseudospermophili E.lateralis E.callospermophili NJ tree

6. Materials and Methods Challenge #1 Collection of quality starting material Collection of quality starting material Identification of a pure sample (one host=one Eimeria species) Identification of a pure sample (one host=one Eimeria species) Repeated sucrose density centrifugation and floats Repeated sucrose density centrifugation and floats Collecting oocysts on the cover slide Collecting oocysts on the cover slide Concentration of oocysts Concentration of oocysts

8. Materials and Methods Challenge #2 Disruption of the wall Disruption of the wall Mechanical Mechanical Glass beadsGlass beads Temperature Temperature Repeated heating/freezing (liquid nitrogen)Repeated heating/freezing (liquid nitrogen) Chemical Chemical NaOCl, H 2 SO 4NaOCl, H 2 SO 4 Treatments unsuitable for wall digestion Treatments unsuitable for wall digestion sonication, ammonia, proteolitic enzymessonication, ammonia, proteolitic enzymes

9. Materials and Methods Successful wall disruption allows for molecular work following standard protocols Successful wall disruption allows for molecular work following standard protocols DNA extraction with proteinase K treatmentDNA extraction with proteinase K treatment Library construction Library construction PCR PCR

10. The future? Requirement to submit sequence data with a description of a new species Requirement to submit sequence data with a description of a new species Creation of a standard list of phylogenetically informative sequences Creation of a standard list of phylogenetically informative sequences

11. Acknowledgments Funding Funding UW INBRE (NIH)UW INBRE (NIH) UW EPSCoR (NSF)UW EPSCoR (NSF) Lab members Lab members Clint OliverClint Oliver Arik SmithArik Smith Dani HofmannDani Hofmann Jason CaseboltJason Casebolt Jason CrouchJason Crouch Stephanie JensenStephanie Jensen