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Jan

Directly from the host: the road map for Onchocerca mitochondrial DNA analysis in Amazonian samples.

The eukaryotic life story cannot be fully reported without reference to the mitochondrion. After a bacterial origin for the mitochondrion was proposed (Margulis 1970), this organelle became one of the tools for a better understanding of eukaryotic evolution. The impact of this symbiotic relationship was enormous for the evolution of life on earth, offering the organisms that have such a “chemical powerhouse” an evolutionary novelty to deal with the complex activities required for survival in an unstable and competitive environment.

For some eukaryotic parasites, the mitochondrion is not always necessary. For instance, in the parasitic protozoa Trypanosoma brucei equiperdum and Trypanosoma brucei evansi the mitochondrial DNA is either partially or completely absent in the bloodstream stage, which means that these two parasites are able to jump from host to host without the need of an insect developmental stage (Lai et al. 2008). This should not be a surprise at all as parasites are both strange and smart creatures and are also limited in their capacity for storage of genetic material for survival in the free living form. Parasites are good examples of survival in hostile environments and have been around for hundreds of millions of years. Even though primitive and parasitic, they demonstrate that an eukaryotic lifestyle is possible without a mitochondrion.

The mitochondrial DNA is also helping us to track the origin and relationships among all eukaryotes (e. g., the use of mitochondrial genes - COXI - to detect human origins and specific mutations to identify organisms - DNA barcoding). Thus, getting access to a complete mitochondrial DNA sequence is worthwhile. Currently, more than 6,600 mitochondrial DNA sequences are stored in the National Center for Biotechnology Information Reference Sequence (ncbi.nlm.nih.gov/genome/organelle/) and this number will increase at a faster rate with the so called New Generation Sequencing technologies (massive parallel DNA sequencing methods represented by sequencing instruments such as the Illumina, 454 Pyroseq, Pacbio, Ion torrent, and, in the near future, the portable “nanopore” sequencing). Parasites that can be cultured in the laboratory are the most prone to be sequenced for their mitochondrial DNA. In general, with the Sanger sequencing or the di-deoxy termination method, mitochondrial DNA sequencing and analysis of a cultured parasite can be considered as an undergraduate final term project, requiring only one person and the routine techniques available in a molecular biology lab. 

However, for parasites that cannot be easily cultivated or isolated in the lab and that are frequently associated with a biological fluid or substrate the situation is different. This is the case of Onchocerca volvulus parasites. Although they can be isolated and cultivated in vitro (with some fine adjustments to the methodology) (Townson & Tagboto 1996), for diagnostic purposes it is highly desirable to analyse them directly from the original sample. If the goal is to obtain precise genetic information (DNA or RNA), the traditional biomolecular techniques do not provide full details. However, for the new sequencing technologies sample composition heterogeneity is not a barrier in getting DNA information. With proper algorithms and controls, parasite and host DNA can be separated and then defined with respect to their nuclear and mitochondrial origin. A report addresses this topic in the Genome Announcements and Highlights section of the January 2016 [111(1)] issue of Memórias do Instituto Oswaldo Cruz: the mitochondrial DNA of O. volvulus from an Amazonian sample (Crainey et al. 2016). The road map: sequence directly from the sample of total DNA with no preliminary isolation steps and pick out complete parasite DNA information. It is getting easier to deal in toto with hidden parasite DNA!

 

Adeilton Alves Brandão | Editor

REFERENCES

Crainey JL, da Silva TRR, Encinas F, Marín MA, Vicente ACP, Luz SLB 2016. The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus. Mem Inst Oswaldo Cruz 111: 79-81.

Lai DH1, Hashimi H, Lun ZR, Ayala FJ, Lukes J 2008. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei. Proc Natl Acad Sci USA 105: 1999-2004.

Margulis L 1970. Origin of eukaryotic cells, Yale University Press, New Haven, 186 pp.

Townson S, Tagboto SK 1996. In vitro cultivation and development of Onchocerca volvulus and Onchocerca lienalis microfilariae. Am J Trop Med Hyg 54: 32-37.

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