Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2005

Thesis Director

Gary Fritz

Thesis Committee Member

Zhiwei Liu

Thesis Committee Member

Gary Bulla

Abstract

Studies on malaria in our "backyard," the Neotropics, have been hampered by the lack of an accurate means of species identification for mosquito vectors of the human malaria parasite (i.e. Plasmodium spp). Mosquitoes in the genus Anopheles are the principle malaria vectors in the Neotropics and are often composed of closely related morphologically similar species complexes. This thesis describes three polymerase chain reaction (PCR) protocols to aid in identifying four anopheline mosquitoes for any life stage, of which three have been incriminated as vectors of human malaria in several locations throughout S. America and might be potential vectors of human malaria in the Chapare and Carrasco valleys of Cochabamba, Bolivia. The first chapter describes a protocol that involves a multiplex PCR with two species specific primers that anneal within the internal transcribed spacer 2 (ITS2) to identify two members of the unresolved Oswaldoi species complex in the subgenus Nyssorhynchus. The second chapter describes a PCR protocol that produces a single amplicon of a section of the ITS2 specific for members of the Albitarsis species complex in the subgenus Nyssorhynchus; digestion of the amplicon with the restriction endonuclease Bfa-I produces two fragments specific to An. marajoara in Bolivia. In addition, I report a new species record for An. marajoara in Bolivia. The third chapter describes a PCR protocol that produces a single amplicon of a section of the ITS2 specific for An. fluminensis, a member of the Arribalzagia Series in the subgenus Anopheles. Examination of ITS2 sequences showed the existence of three short repeat units (aprox. 125 bp) within the ITS2 of An.fluminensis. This study also records the presence of An. fluminensis for the first time in Cochabamba, Bolivia. The PCR protocols described in this thesis, then, will simplify the task of identifying four anopheline species and facilitate studies that aim to elucidate their basic biology, ecology, and behavior.

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