Browsing by Autor "J. Russell Stothard"

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A preliminary survey of mitochondrial sequence variation in Triatominae (Hemiptera: Reduviidae) using polymerase chain reaction-based single strand conformational polymorphism (SSCP) analysis and direct sequencing
(Cambridge University Press, 1998) J. Russell Stothard; Yutaka Yamamoto; A. Cherchi; Ana Lineth García; Sebastião Aldo S. Valente; Christopher J. Schofield; Michael A. Miles
Abstract Genetic variation within triatomine bugs was investigated by amplification of a 400 bp portion of the mitochondrial 16S ribosomal RNA gene by polymerase chain reaction (PCR), using evolutionarily conserved primers, from a selection of species representative of the genera Rhodnius, Triatoma and Panstrongylus . Amplification products were subsequently screened for sequence variation using single strand conformational polymorphism analysis (SSCP) and also subjected to direct sequencing. Single strand conformational polymorphism analysis could detect variation within and between genera; intraspecific variation was also detected and SSCP profiles appear to be useful for identification purposes at the inter- and intraspecific levels. A 290 bp multiple alignment of 15 sequences obtained from nine species was generated, phylogenetic inference subsequently used three methods; a distance estimate, maximum parsimony and maximum likelihood. This 16S region exhibited considerable variation which ranged from intergeneric to intraspecific levels. Phylogenies from these three methods of inference were in broad agreement. Triatoma and Panstrongylus were more closely related to each other than either was to Rhodnius , in keeping with the current taxonomic appraisal.
Random Amplification of Polymorphic DNA as a Tool for Taxonomic Studies of Triatomine Bugs (Hemiptera: Reduviidae)
(Oxford University Press, 1998) A García; H. Carrasco; Christopher J. Schofield; J. Russell Stothard; Iain A. Frame; Sebastião Aldo S. Valente; Michael A. Miles
Eleven of 27 decameric primers were found to be suitable for random amplification of polymorphic DNA (RAPD) from triatomine bugs on the basis that they produced discrete profiles and distinguished among Panstrongylus megistus (Burmeister), Rhodnius prolixus Stål, and Triatoma infestans (Klug). The legs, or single leg segments, of individual bugs were used as the source of DNA so that the taxonomic value of the bug was conserved. Within the scope of the specimens studied, RAPD profiles allowed assignment to species even when bugs were kept dry for up to 12 mo. Profiles for individuals within a species were not identical. RAPD profiles, with the specimens tested, distinguished among species of 3 pairs considered to be morphologically similar and closely related, namely, Rhodnius ecuadorensis Lent & León and Rhodnius pictipes Stål; Rhodnius nasutus Stål, and Rhodnius neglectus Lent; Rhodnius prolixus Stål and Rhodnius robustus Larrousse. RAPD data conformed with the perceived affinities among these species. RAPD polymorphisms were seen with T. infestans from 3 different localities, but none of the polymorphisms was confined to 1 source. RAPD provided a molecular basis to reassess taxonomic relationships within the Triatomine subfamily. The accurate distinction of triatomine species and of intraspecific bug populations may contribute to elimination of vector-borne Chagas disease from the Americas.