Laser microdissection microscopy and single cell PCR of avian hemosporidians.

Avian malaria parasites (Plasmodium spp.) and related species of Haemoproteus constitute a remarkably diverse and species rich group of blood parasites. Analyses of the mitochondrial cytochrome b gene of these hemosporidians have demonstrated unexpected patterns of host distribution, host shifts, and host sharing. However, deeper insights into these patterns require access to multiple genetic markers and genetic analyses of single parasite cells. In the present study, we demonstrate the potential of laser microdissection microscopy (Olympus/MMI CellCut microdissection system) for solving these 2 problems. This technique was used for isolation of single blood stages and ookinetes of avian Haemoproteus and Plasmodium spp., which were then successfully used for DNA isolation, amplification, and sequencing. The methods of single cell dissection of hemosporidian parasites and PCR-based analyses with dissected single cells are described. These methods can be used to isolate substantial quantities of pure hemosporidian parasite DNA for large-scale sequencing, essential information when designing primers for developing multiple nuclear genetic markers. Such markers can then be applied to isolated single parasite cells for identification of parasites in mixed infections and deciphering mechanisms behind apparent reproductive isolation between parasite lineages. This method can be used in the molecular investigation of blood parasites of birds, reptiles, and fish because it enables removing the parasite DNA from the overpowering host DNA, which is present in red blood cells.