Using Molecular Analysis to Using Molecular Analysis to Examine the Phylogeny of Examine the Phylogeny of

Symbiotic Water Mites in Two Symbiotic Water Mites in Two Subgenera:Subgenera:

UnionicoloidesUnionicoloides and and ParasitaxParasitax

Dan DeatherageDan Deatherage

Kevin MyersKevin Myers

September 3,2004September 3,2004

BackgroundBackground

Symbiotic water mitesSymbiotic water mites

Mussels provide hosts to mitesMussels provide hosts to mites

Size requires molecular techniquesSize requires molecular techniques

Molecular phylogeny Vs. Classical Molecular phylogeny Vs. Classical PhylogenyPhylogeny

Cytocrome Oxidase I (COI) gene Vs. Cytocrome Oxidase I (COI) gene Vs. Internal Transcriber Sequence (ITS)Internal Transcriber Sequence (ITS)

Why These Mites?Why These Mites?Readily found and collectedReadily found and collectedGenerally many individuals in each Generally many individuals in each populationpopulationMites similar to these have been heavily Mites similar to these have been heavily studiedstudiedThe COI sequences for these mites are The COI sequences for these mites are not knownnot knownThis field of study is open to new research This field of study is open to new research without fear of being “scooped”without fear of being “scooped”The dissemination of species was begun The dissemination of species was begun by Dr. Edwardsby Dr. Edwards

Unionicola sp.•All species of Unionicola are morphologically very similar

Utterbackita imbecillis Anadonta suborbiculata

Proposed ResearchProposed Research

Project begun through the UExplore Project begun through the UExplore Undergraduate Research Program Undergraduate Research Program summer 2004.summer 2004.

Mites chosen because their close classical Mites chosen because their close classical phylogeny classification.phylogeny classification.

Results during the summer have shown Results during the summer have shown that the methods we will use are effective.that the methods we will use are effective.

Species of Mites to Study Species of Mites to Study Include:Include:

From the subgenus From the subgenus Parisitax:Parisitax:– Unionicola formosaUnionicola formosa from from Pyganadon cataractaPyganadon cataracta– U. formosa U. formosa from from P. grandisP. grandis

From the subgenus From the subgenus UnionicoloidesUnionicoloides::– Unionicola kavanaughiUnionicola kavanaughi– U. gailaeU. gailae– U. hoeseiU. hoesei– U. lasalleiU. lasallei– U. amanditaU. amandita

Stored Mites &Predetermined

Primers

For Each Mite

Primer Design with NetPrimerPrimer Design with NetPrimer

Stored Mites &Predetermined

Primers

PCR ExtractedDNA

For Each Mite

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.

Stored Mites &Predetermined

Primers

Sequence PurifiedPCR Product

PCR ExtractedDNA

For Each Mite

CodonCode AlignerCodonCode Aligner

Stored Mites &Predetermined

Primers

Sequence PurifiedPCR Product

Create a MolecularPhylogeneic Tree

PCR ExtractedDNA

For Each Mite

ClustalWClustalW

Molecular Phylogenic TreeMolecular Phylogenic Tree

TimelineTimeline

Sept 15Sept 15 Preliminary extraction underway. All Preliminary extraction underway. All materials materials gathered. gathered. Sept 30Sept 30 All DNA extracted from initial mites and 2 All DNA extracted from initial mites and 2

species sent for sequencing.species sent for sequencing.Oct 15Oct 152 more species sequenced, initial 2 species 2 more species sequenced, initial 2 species

sequence analysis began.sequence analysis began.Oct 30Oct 30Final 2 sequences returned, analysis Final 2 sequences returned, analysis

continuing.continuing.Nov 15Nov 15 All sequence analysis complete, All sequence analysis complete, manuscript manuscript begun.begun.Nov 30Nov 30 All lab work done, and manuscript in final All lab work done, and manuscript in final

stages.stages.Dec 15Dec 15 Manuscript submitted for class and for Manuscript submitted for class and for

publication.publication.

BudgetBudget

ItemItem CostCost

DNA Sequencing DNA Sequencing $300$300

(16 sequences)(16 sequences)

Qiagen DNeasy Tissue KitQiagen DNeasy Tissue Kit $156$156

Promega PCR Master Mix Promega PCR Master Mix $67 $67 (100 reactions)(100 reactions)

Total: $523Total: $523

Grade AgreementGrade Agreement

Level of Progress AchievedLevel of Progress Achieved Points ReceivedPoints Received

DNA successfully extracted DNA successfully extracted 3.03.0

PCR product successfully amplified PCR product successfully amplified 4.04.0

Sequence data successfully Sequence data successfully returned returned 1.51.5

Sequence data successfully Sequence data successfully analyzed analyzed 4.04.0

Total Points for Each Mite Species Total Points for Each Mite Species 12.512.5

Remaining Happy Throughout the Remaining Happy Throughout the Semester Semester PricelessPriceless

ReferencesReferences

Boore, J. L. 1999. Animal Mitochondrial Genomes. Boore, J. L. 1999. Animal Mitochondrial Genomes. Nucleic Acids Research, 27(8):1767-1780.Nucleic Acids Research, 27(8):1767-1780.Edwards, D. D. and Ernsting, B. R. 2004. UExplore Edwards, D. D. and Ernsting, B. R. 2004. UExplore Undergraduate Research Proposal.Undergraduate Research Proposal.Myers, Kevin, Eyler, Andrea, and Rasure, Mark. 2004. Myers, Kevin, Eyler, Andrea, and Rasure, Mark. 2004. Unpublished Data.Unpublished Data.Navajas, M. and P. Boursot. 2003. Nuclear ribosomal Navajas, M. and P. Boursot. 2003. Nuclear ribosomal DNA monoplyly versus mitochondrial DNA polyphyly in DNA monoplyly versus mitochondrial DNA polyphyly in two closely related mite species: the influence of life two closely related mite species: the influence of life history and molecular drive. Proceedings of the Royal history and molecular drive. Proceedings of the Royal Society of London B Biological Sciences, 270 Suppl Society of London B Biological Sciences, 270 Suppl 1:S124-127.1:S124-127.

Questions? Ideas?Questions? Ideas?