ORIGINAL PAPER M. Vobis Æ J. D’Haese Æ H. Mehlhorn Æ N. Mencke B. L. Blagburn Æ R. Bond Æ I. Denholm Æ M. W. Dryden P. Payne Æ M. K. Rust Æ I. Schroeder Æ M. B. Vaughn D. Bledsoe Molecular phylogeny of isolates of Ctenocephalides felis and related species based on analysis of ITS1, ITS2 and mitochondrial 16S rDNA sequences and random binding primers Received: 1 July 2004 / Accepted: 19 July 2004 / Published online: 1 September 2004 Ó Springer-Verlag 2004 Abstract The phylogenetic relationships among 31 dif- ferent flea isolates representing seven different species were studied by nucleotide sequence comparison of the internal transcribed spacer 1 (ITS1), internal transcribed spacer 2 (ITS2) and/or mitochondrial 16S ribosomal RNA gene (mt16S-rDNA) to examine the patterns of variation. Results show that all regions are useful in discriminating among flea species. In Ctenocephalides felis and Tunga penetrans, some differences in these gene regions occurred among different isolates within the same species. In the latter case, the differences are in the mt16S-rDNA region, with one isolate showing 48% divergence in nucleotide sequence. The taxonomic implications of this result are unclear at present. The gene regions revealed differences between C. felis isolates only after DNA sequencing the PCR products. Further differentiation among C. felis isolates was obtained using four different random binding primers (decamers) and primers for mammalian aldolase to amplify narrow differences in the genome. Using these primers we were able to discriminate between different C. felis isolates and determine that some of the genetic variation coin- cided with minor differences in response to the control agent imidacloprid. However, overall findings do not support the existence of subspecies of C. felis. Introduction Genus and species determination of fleas is generally based on a variety of morphological criteria (Lane and Crosskey 1993; Kra¨mer and Mencke 2001; Mehlhorn 2001). However, little is known of the intra-specific systematics of these species, and in particular whether four proposed subspecies of C. felis have any biological relevance (Hopkins and Rothschild 1953). Intraspecific taxa may be differentially sensitive to repellents and/or insecticides and thus may be important when evaluating insecticidal efficacy or status of insecticide resistance. To determine if such differences exist, numerous isolates (Table 1) were examined using different molecular markers. The internal transcribed spacer regions 1 and 2 (ITS1/ITS2) separate nuclear ribosomal genes and have proven useful for analysing genetic relationships among species (Baldwin et al. 1995). These regions vary to different degrees among species, and have also been used to differentiate populations within a species, e.g. mites (Essig et al. 1999), nematodes (Heise et al. 1999) or heteropteran bugs (Marcilla et al. 2002). Constructing phylogenetic trees by nucleotide sequence comparison is not restricted to these regions, M. Vobis Æ J. D’Haese Æ H. Mehlhorn (&) Institute for Zoomorphology, Cell Biology und Parasitology, Heinrich-Heine University, 40225 Du¨sseldorf, Germany E-mail: mehlhorn@uni-duesseldorf.de N. Mencke Æ I. Schroeder Animal Health Division, Bayer HealthCare, 51368 Leverkusen, Germany B. L. Blagburn Auburn University, Auburn, AL, USA M. B. Vaughn Æ D. Bledsoe Animal Health Division, Bayer HealthCare, Shawnee Mission, KS, USA I. Denholm Rothamsted Research, Harpenden, UK M. W. Dryden Æ P. Payne Kansas State University, Manhattan, KS, USA M. K. Rust University of California, Riverside, CA, USA R. Bond Royal Vet College, London, UK Parasitol Res (2004) 94: 219–226 DOI 10.1007/s00436-004-1201-x