Combination of cytochrome b heteroduplex-assay and sequencing for identification of triatomine blood meals

• Published in: Infection, genetics and evolution genetics and evolution, 2012-01, Vol.12 (1), p.21-27
• Main Authors: Buitrago, Rosio, Depickère, Stéphanie, Bosseno, Marie-France, Patzi, Edda Siñani, Waleckx, Etienne, Salas, Renata, Aliaga, Claudia, Brenière, Simone Frédérique
• Format: Article
• Language: English
• Subjects: Animal biology; Animals; Biochemistry, Molecular Biology; Biodiversity and Ecology; Biological and medical sciences; birds; Blood; Blood meal; color; Cytochrome b; Cytochromes b - genetics; DNA; DNA Fragmentation; DNA Primers; Ecology, environment; electrophoresis; Environmental Sciences; Epidemiology. Vaccinations; General aspects; genes; Heteroduplex; Heteroduplex Analysis; hosts; Infectious diseases; intraspecific variation; Invertebrate Zoology; Life Sciences; mammals; Medical sciences; microorganisms; Molecular biology; polymerase chain reaction; rearing; Reproducibility of Results; reptiles; screening; Sensitivity and Specificity; Sequence Analysis, DNA; Sequencing; species identification; Species Specificity; Symbiosis; Triatominae; Triatominae - chemistry; Triatomines
• ISSN: 1567-1348; 1567-7257
• DOI: 10.1016/j.meegid.2011.09.005

► We validate PCR cytochrome b heteroduplex assay to identify blood meals in triatomines. ► Blood meals detection provides information on triatomine movements among ecotopes. ► This method combined with sequencing is a useful tool in epidemiological studies. The identification of blood meals in vectors contributes greatly to the understanding of interactions between vectors, microorganisms and hosts. The aim of the current work was to complement the validation of cytochrome b (Cytb) heteroduplex assay (HDA) previously described, and to add the sequencing of the Cytb gene of some samples for the identification of blood meals in triatomines. Experimental feedings of reared triatomines helped to clarify the sensitivity of the HDA. Moreover, the sequencing coupled with the HDA, allowed the assessment of the technique’s taxonomic level of discrimination. The primers used to produce DNA fragments of Cytb genes for HDA had a very high sensitivity for vertebrate DNAs, rather similar for mammals, birds and reptiles. However, the formation of heteroduplex depended on blood meal’s quality rather than its quantity; a correlation was observed between blood meals’ color and the positivity of HDA. HDA electrophoresis profiles were reproducible, and allowed the discrimination of blood origins at the species level. However, in some cases, intraspecific variability of Cytb gene generated different HDA profiles. The HDA based on comparison of electrophoresis profiles is a very useful tool for screening large samples to determine blood origins; the subsequent sequencing of PCR products of Cytb corresponding to different HDA profiles allowed the identification of species whatever the biotope in which the vectors were captured.