Analysis of the 5.8S rRNA Gene and the Internal Transcribed Spacers in Naegleria spp. and in N. fowleri

Abstract

Internal transcribed spacers (ITS) and the 5.8S ribosomal gene of 21 Naegleria fowleri strains and eight other species including Naegleria gruberi were sequenced. The results showed that this region can help differentiate between and within species. The phylogeny of Naegleria spp. deduced from the ITS and the 5.8S gene produced four major lineages, fowleri-lovaniensis, galeacystis-italica-clarki-gruberi-australiensis, andersoni-jamiesoni, and pussardi, that fit perfectly with those inferred from the 18S rRNA gene analysis. The N. gruberi isolate, NG260, was closely related to Naegleria pussardi. The other N. gruberi isolates branched together with Naegleria australiensis in another lineage. The ITS and 5.8S results for N. fowleri were congruent with those previously deduced by RAPD analysis. The phylogenetic analysis inferred from ITS and RAPD data revealed two major groups. The French Cattenom and Chooz and South Pacific strains constituted the first group. The second group encompassed the strains corresponding to the Euro-American and Widespread RAPD variants and shared the same substitution in the 5.8S gene. In addition, it was possible to define species specific primers in ITS regions to rapidly identify N. fowleri.

Rapid, Sensitive, and Discriminating Identification ofNaegleriaspp. by Real-Time PCR and Melting-Curve Analysis

“…For example, the observation that N. fowleri, which has the most AT-rich ITS1 in the genus (6), also has the most significant leftward (low temperature) peak suggests that this peak may correspond to (or incorporate or overlap with) ITS1. We postulate that the currently recognized N. fowleri genotypes that vary in the number of tandem repeats in ITS1 (6,20,33) might be distinguishable in this part of the melting curve. We intend to pursue the theoretical interpretation of the melting curves reported here and to explore the identification of the N. fowleri genotypes and additional Naegleria species by similar criteria.…”

Multigene phylogenies of clonal Spumella-like strains, a cryptic heterotrophic nanoflagellate, isolated from different geographical regions

“…Lajeunesse, 2005). Thus, this highly variable locus has attracted attention as a potential marker for the resolution of closely related taxa in different protists (Diggles & Adlard, 1997;Katz et al, 2004;Pelandakis et al, 2000;van Hoek et al, 1998). Also, the protein-coding genes we evaluated in this study (actin, alpha-and beta-tubulin) are known as fast-evolving in a range of different protist taxa (Kim et al, 2004;Nishi et al, 2005).…”

Fish-isolated Naegleria strains and their phylogeny inferred from ITS and SSU rDNA sequences

“…However, the evolutionary conserved nature of these genes that makes them relatively uninformative at the generic and species levels in eukaryotes has been stressed several times (Sogin 1991, Coleman et al 1998, Sogin and Silberman 1998, Pélandakis et al 2000. The spacer regions of nuclear ribosomal genes have generally been found more variable and useful in comparing closely related species or populations (Felleisen 1997, De Jonckheere 1998, Pélandakis et al 2000.…”

“…Because of a rather uniform morphology, the introduction of molecular approaches became of fundamental importance in the history of species definitions within the genus Naegleria Alexeieff, 1912 (De Jonckheere 1994, 1998, 2002, 2004, Pélandakis et al 2000.…”