High mitochondrial sequence divergence in synanthropic flea species (Insecta: Siphonaptera) from Europe and the Mediterranean

Background: Adult fleas are haematophagous ectoparasites of warm-blooded vertebrates, particularly mammals. Among them, the cat flea (Ctenocephalides felis) and the human flea (Pulex irritans) have high veterinary-medical significance, owing to their cosmopolitan distribution and role in the transmission of important vector-borne pathogens. While the taxonomy of Ct. felis has been investigated on a morphological basis during the past decades, its molecular-phylogenetic analyses have been only recently conducted. This study expands the knowledge on Ct. felis from hitherto less studied geographical regions, and includes representatives from additional flea families, less investigated with molecular approaches. Methods: Fleas were collected in four countries of the Mediterranean Basin (Croatia, Italy, Malta and Israel), as well as in Hungary, from domestic and wild carnivores, rodents and humans. The DNA extracts of representative fleas (n = 148), belonging to ten species of eight genera, were used for PCR amplification of part of their cytochrome c oxidase subunits 1, 2 (cox1, cox2) and 18S rRNA genes, followed by sequencing and phylogenetic analyses. Results: The majority (65.6%) of Ct. felis felis cox2 sequences showed 99.4-100% similarity to each other (haplogroup A), whereas those from Malta and Israel had 98.1-98.7% sequence similarity (haplogroup B), and a third sequence from Israel (haplotype C) had as low as 96.3% sequence similarity in comparison with a reference sequence from group "A". Except for the shape of the head, no consistent morphological differences (e.g. in chaetotaxy) were found between haplogroups "A" and "C". Haplotypes of Ct. canis were genetically more homogenous, with 99.6-100% sequence similarity to each other. However, when P. irritans collected from humans was compared to those from three species of wild carnivores, these only had 96.6% cox2 similarity. The mouse flea, Leptopsylla segnis and the northern rat flea, Nosopsyllus fasciatus were both shown to have haplotypes with low intraspecific cox2 similarities (96.2 and 94.4%, respectively). Taken together, differences between mitochondrial lineages within four flea species exceeded that observed between two Chaetopsylla spp. (which had 97.3% cox2 similarity). The topologies of cox1 and cox2 phylogenetic trees were in line with relevant sequence comparisons. Conversely, 18S rRNA gene analyses only resolved differences above the species level. Conclusions: Ctenocephalides felis felis, P. irritans, L. segnis and N. fasciatus were shown to have such a high level of mitochondrial gene heterogeneity, that the uniformity of these flea taxa should be reconsidered. Although the present results are limited (especially in the case of L. segnis and N. fasciatus), there appears to be no geographical or host restriction, which could explain the divergence of these genetic lineages.