Ixodes ricinus ticks have a functional association with Midichloria mitochondrii .

In addition to being vectors of pathogenic bacteria, ticks also harbor intracellular bacteria that associate with ticks over generations, aka symbionts. The biological significance of such bacterial symbiosis has been described in several tick species but its function in is not understood. We have previously shown that ticks are primarily inhabited by a single species of symbiont, , an intracellular bacterium that resides and reproduces mainly in the mitochondria of ovaries of fully engorged females. To study the functional integration of into the biology of , an -depleted model of ticks was sought. Various techniques have been described in the literature to achieve dysbiosed or apo-symbiotic ticks with various degrees of success. To address the lack of a standardized experimental procedure for the production of apo-symbiotic ticks, we present here an approach utilizing the membrane blood feeding system. In order to deplete from ovaries, we supplemented dietary blood with tetracycline. We noted, however, that the use of tetracycline caused immediate toxicity in ticks, caused by impairment of mitochondrial proteosynthesis. To overcome the tetracycline-mediated off-target effect, we established a protocol that leads to the production of an apo-symbiotic strain of , which can be sustained in subsequent generations. In two generations following tetracycline administration and tetracycline-mediated symbiont reduction, was gradually eliminated from the lineage. Larvae hatched from eggs laid by such -free females repeatedly performed poorly during blood-feeding, while the nymphs and adults performed similarly to controls. These data indicate that represents an integral component of tick ovarian tissue, and when absent, results in the formation of substandard larvae with reduced capacity to blood-feed.