The 2005 ESA Annual Meeting and Exhibition
December 15-18, 2005
Ft. Lauderdale, FL

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Friday, December 16, 2005
D0195

Simultaneous use of insect tissues for DNA and morphological studies

Catherine Duckett, duckett.catherine@si.nmnh.edu, Smithsonian Institution, Entomology, NHB MRC105, P.O. Box 37012, Washington, DC and Scott Whittaker, whittaks@si.edu, Smithsonian Institution, Laboatories of Analytical Biology, MRC 104, Washington, DC.

"Find'em and Grind'em" has been, if not a credo of molecular entomologists, it has at least been ascribed to them by morphologists. Numerous entomologists using molecular techniques have asserted that exoskeltal material does not need to be damaged in the extraction process. Imaging of chitinized insect structures to demonstrate their utility for morphological study after DNA extraction has not been done until this year, (for lepidopteran genitalia). Here we show scanning electron and light micrographs of insects which were either intact, perforated or partially dissected when extracted; comparisons with unextracted control taxa are made. Extraction enzymes do not damage the vast majority of tissues and taxa tested. Conversely, current extraction enzymes and buffers leave chitinized structures very clean for SEM analysis and if tissue and enzyme quantites are correctly calibrated perfectly clean preparations of mouthparts and genitalia can be obtained. DNA was sequenced to confirm efficacy of the techniques; ease of PCR amplification of multicopy genes depending on collection techniques and degree of dissection during extraction is discussed. DNA extractions that yielded clean sequences were obtained from insects collected with a variety of collection and preservation techniques commonly used by morphologists these include: killing w/ethyl acetate for short periods, fixing larvae with boiling water, and use of 70% isopropanol (drugstore grade). Sequenceable DNA was also obtained from insects caught in tropical trapping programs and dried specimens using these methods.



Species 1: Coleoptera Chrysomelidae Alagoasa sp
Species 2: Hymenoptera Formicidae
Species 3: Hemiptera Membracidae
Keywords: SEM imaging, DNA extraction