Title:Molecular investigation of ectoparasites from Malta
Malta, owing to its unique geographical location, offers a suitable environment to study the genetic connection or isolation of synanthropic ectoparasites, in comparison with those from nearby or more distant countries. With this aim, during the present study, non-flying ectoparasites have been collected on 24 locations in Malta, followed by morphological and molecular analyses. These ectoparasites included: 540 Rhipicephalus sanguineus ticks, one Hyalomma lusitanicum tick, 42 Ctenocephalides felis fleas, 47 Xenopsylla cheopis fleas, 20 Cimex lectularius bedbugs, 38 Felicola subrostratus lice, 17 Laelaps echidninus and 42 L. hilaris mites. Immature stages of R. sanguineus occurred significantly more frequently on cats than on dogs. Significantly more larvae and nymphs of this tick species were collected from female dogs and female cats in comparison with males. Concerning molecular analyses in a phylogeographical context, the amplified part of the cytochrome c oxidase (CO) subunit 1 (COI) and 16S rRNA genes of R. sanguineus ticks collected from cats in Malta showed 100% sequence identity with each other and with samples from Italy. Similarly, F. subrostratus lice collected from cats in Malta showed 100% COI sequence identity with each other and samples from Hungary or those reported from the USA. On the other hand, the CO subunit 2 (COII) gene of Ct. felis fleas collected from dog and cat were heterogenous within Malta, and had 99.9% sequence similarity with samples from Hungary, 99.9%-100% sequence similarity with samples from New Zealand, and lower degrees of similarity with samples from other countries of the Old World. The COI sequence of Hy. lusitanicum tick from rabbit, Malta, was 99.8% identical with that of samples from the western Mediterranean Basin. The COI sequence of Ci. lectularius (bedbug) specimens had 99.7-100% sequence similarity within Malta, and phylogenetically clustered closest to bedbugs from the Czech Republic, and more distantly from samples collected in Italy. Concerning molecular screening of pathogens, 38.5% of Ct. felis fleas were positive for Rickettsia felis, with one specimen containing Mycoplasma haemofelis-like DNA. In conclusion, because R. sanguineus is an endophilic ticks species (preferring to live in smaller spaces or even indoors), its immature stages occur more likely on pet animals using such sheltered environments more frequently or for a prolonged time. Varying intraspecific genetic similarities between ectoparasites from Malta and those from other countries most likely reflect differences between such characters of the studied ectoparasite categories, which influence gene flow between their populations. These include host specificity, duration of stay on host, transportation of hosts or transportation of items where they develop. Hyalomma lusitanicum and L. hilaris are new records for the ectoparasite fauna of Malta. This is also the first detection of R. felis and a feline haemoplasma in Malta.