Abstract
Background: Naegleria are abundant, free-living, freshwater amoebae with a worldwide distribution that are able to differentiate into swimming flagellates. Species within this genus are quite diverse, probably because the genus evolved over a billion years ago. Mesophilic species like N. gruberi are found on the five temperate continents, while N. antarctica are thermophobic, and N. fowleri are thermophilic opportunistic human pathogens (the brain-eating amoebae). Previous analysis of short ribosomal ITS sequences catalogued roughly 40 geographically separated species. Our analysis, using a combination of Closed- Tube Barcoding of the COI gene target plus DNA sequencing, has improved the definition of species. Analysis of about 75 clonal isolates from around the world using both COI and ITS sequences show agreement in the degree of relatedness among isolates in most cases. Results: Closed-Tube DNA Barcoding is an efficient, cost-effective method for amplifying the COI barcoding target sequence from large numbers of isolates and then scanning the resulting single-stranded DNA for sequence variations using Lights-On/Lights-Off probes. In order to resolve all species within this genus, we use a universal set of nine probes having three subsets. Subsets are labeled in different fluorescent colors. This experimental design allows us to compare the fluorescent signatures of different isolates and immediately observe whether sequence differences are clustered in one region of the COI target, or distributed throughout. These predictions can be confirmed by using Dilute-N-Go sequencing. Significance: Our ground-breaking study of Naegleria provides an affordable method for characterization of species and species variation within microscopic eukaryotes--a world that is largely unknown. Using this approach we will be able to map species distributions in small or large ecosystems. We can also use our approach to selectively test for N. fowleri, or virtually any pathogen in a water sample. Supported by Brandeis University.