Masters Theses

Sunitha Vege

Date of Award

12-2000

Degree Type

Thesis

Degree Name

Master of Science

Major

Ecology and Evolutionary Biology

Major Professor

Gary F. McCracken

Committee Members

Susan E. Riechert, Steven W. Wilhelm

Abstract

Primers for PCR (Polymerase Chain Reaction) amplification of targeted DNA sequences were developed for the corn earworm (Helicoverpa zea) and tobacco budworm (Heliothis virescens), both major agricultural insect pests that are eaten by bats. Two H. zea primer sets from an intron region of the nuclear PBAN locus (Davis et al., 1992) showed species-specific amplification when tested against genomic DNA of H. zea, H. virescens, Spodoptera exigua, and S. frugiperda. H. virescens mitochondrial (Miller et al., 1990) and nuclear (Logsdon et al., 1995) primers were species-specific when tested against genomic DNA of H. virescens, H. zea, S. exigua, and S. frugiperda. Molecular techniques were applied to fecal material for identifying these moths in DNA extracted from feces of bats. Feces were obtained through controlled feedings of target moth species to big brown bats, Eptesicus fuscus. Species-specific DNA markers for H. zea and H. virescens were tested against fecal DNA of known insect content. Four fecal extraction methods, modified DNeasy, N-phenacylthiazolium bromide (PTE), guanidium thiocynate (GuSCN/silica), and modified sodium lysis were assessed in their abilities to yield good quality DNA from bat feces. The modified DNeasy protocol yielded the most consistent PCR amplification results with the moth DNA markers. Although the markers were species-specific for genomic moth DNA, the markers were not species-specific for moth DNA extracted from fecal material, with PCR products of the expected sizes amplifying against fecal DNA for the target and non-target moth species. Fecal DNA isolated using the PTE, GuSGN/silica, and modified sodium lysis protocols gave poor PCR amplification results. Contaminants and inhibitors present in the fecal DNA were suspected to interfere with PCR. PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) was tested to differentiate between specific and non-specific PCR products that amplified from fecal DNA. Fecal PCR products for the target and non-target moth species were all cut with the same restriction enzymes. Therefore, PCR-RFLP was not useful for distinguishing between moth species in bat fecal DNA. In a separate experiment, big brown bats were genotyped from their feces using three microsatellite primers (EF1, EF6, and MM5). Genotypes obtained from bat fecal DNA consistently matched the genotypes obtained from DNA extracted from wing membrane tissue of the same bats. Genotypes obtained from multiple fecal DNA samples from the same bat were identical. Fecal studies present research opportunities for addressing ecological and behavioral questions for animals that are difficult to capture, rare, or endangered. Unlike tissue material, feces can be collected without the need of CITES permits.

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