Molecular screening for Midichloria bacteria in hard and soft ticks (acari: Ixodida)

Ticks can harbour microbial communities that play different roles in the biology of the host. Among these bacteria, Midichloria mitochondrii (Order Rickettsiales; family Midichloriaceae) was firstly described inside the intermembrane space of mitochondria of Ixodes ricinus females oocytes and subsequently observed in salivary glands. This bacterium is present in 100% of females and immatures, is vertically transmitted and is less prevalent in males (44%). In addition, the possibility of horizontal transmission through the blood meal is suggested by serological and molecular analyses showing positivity of mammalian bloods and sera to M. mitochondrii circulation. However, the role of this bacterium is still unknown. Several species belonging to the six most important genera of hard ticks (i.e. Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Rhipicephalus) have been observed to harbour Midichloria bacteria, but only I. holocyclus presents bacterial loads similar to I. ricinus. This work aims to expand the knowledge about the distribution of Midichloria bacteria in ticks, screening a total of 93 samples (16 Ixodidae and 1 Argasidae species) from different geographical areas by using qualitative (targeting the 16S-rRNA) and quantitative (targeting the gyrB gene) PCR approaches. A total of 8 species out of 16 hard tick species were found positive for Midichloria at different inter-intraspecific prevalence levels. A 100% prevalence observed in I. aulacodi females and a high Midichloria load could indicate a mutualistic relationship. The obtained 16S-rRNA sequences showed between 99% and 100% similarity with M. mitochondrii, and, basing on a phylogenetic analysis, we propose that these bacteria could be classified within the genus Midichloria. Furthermore, our results support the possibility of a horizontal transmission of these bacteria, as it often happens for tick-borne rickettsiae. Further studies focused on other genetic markers will allow to understand the genetic variability of Midichloria in ticks and to test the above hypotheses.