Localization and quantification of intracellular symbiont Midichloria mitochondri in organs of Ixodes ricinus ticks

Midichloria mitochondrii is an intracellular bacterium member of the recently described family of the Rickettsiales, Candidatus Midichloriaceae, that encompasses a wide group of intracellular bacteria associated with ticks (Montagna et al., 2013, Appl Environ Microbiol., 79(10):3241-8). This bacterium has been found in different species of ticks; in particular, in Ixodes ricinus tick, it has been detected in 100% of the examined I. ricinus females across its geographical distribution and in about 50% of males. M. titochondrii has been observed not only in the cell cytoplasm, but also inside the mitochondria, within the intermembrane space (Sacchi et al, 2004, Tissue Cell., 36(1):43-53). Up to now, we know that this symbiont is present in the ovary and salivary glands of the I. ricinus tick (Mariconti et al, 2012, Microbiology, 158(Pt 7):1677-83; Epis et al., 2013, Ticks Tick Borne Dis., 4(1-2):39-45) but no studies have so far investigated whether M. mitochondrii is spread and quantified in other organs of the tick. In particular, taking in consideration indirect evidences that suggest that the bacterium could be transmitted to vertebrates during the blood meal of the tick, the analysis of salivary glands should be of extremely interest. Moreover the localization of M. mitochondrii in the body of the arthropod and its ability to diffusion are unknown. In this work we investigated the localization/quantification of M. mitochondrii in the body of the tick using immunofluorescence assay with specific anti-FLID antibody for M. mitochiondri detection and quantitative Real-Time PCR in different organs of I. ricinus: Two genes were amplified: the gene of M. mitochondrii gyrB coding for gyrase B and the nuclear gene of I. ricinus cal coding for the protein Calreticulin. Adult female ticks both “semi-engorged” (i.e. a tick at half of its blood meal) and “non-engorged” (i.e. a tick that didn’t start its blood meal yet) were analyzed. M. mitochondrii was detected in different body organs of the ticks. Furthermore differences in bacterial burden between the two analyzed life cycle period of the tick were found. In fact an increase of M. mitochondrii burden, such as one of classical pathogens transmitted by the tick, can be noticed during the meal in salivary glands and rostrum. This study thus offered further evidences that M. mitochondrii is transmitted by ticks to vertebrates through saliva. Interestingly each trachea of the analyzed I. ricinus was positive for M. mitochondrii: this suggest that the respiratory tract can be used by M. mitochondrii as a dissemination way in the tick. This datum was described in previous works for organisms belonging to Rickettsia genus; Baldridge and colleagues (2007, J Invertebr Pathol., 94(3):163-74) experimentally infected tick of the specie Ixodes scapularis (a tick with American diffusion) with a cultivable Rickettsia monacensis able to produce the green fluorescence protein (GFP). After experimental infection, fluorescent R. monacensis were found distributed in the respiratory system of ticks; this result allowed the researchers to consider the tracheal system as a way of dissemination of bacteria and a possible "reservoir" during moulting. Similarly, Midichloria may also use the respiratory system as a way to spread within the I. ricinus tick.