Resumo

The oviposition is a vulnerable stage of the tick life cycle. Rhipicephalus(Boophilus) microplus, as all Ixodidae and Argasidae, has a specialized gland, the Gene’s organ, which produce a wax that is smeared on egg surface during oviposition. Rhipicephalus microplus lays eggs in the soil near the roots of grass, or in similar highly moist environments that are prone to biofilm formation. However, as the eggs remain viable and show no visible signs of microbial colonization, we hypothesized that the egg coating might have anti-biofilm properties. By scanning electron microscopy we observed that Pseudomonas aeruginosa is unable to form a biofilm on the surface of intact tick eggs which ended up harboring only a few bacterial cells. Conversely, biofilm form readily on eggs lacking their wax coatings. We show here that the coating inhibits biofilm formation by both Gram-negative and Gram-positive bacteria, though by different mechanisms. Using several spectroscopic techniques (1H and 13C NMR, ESI-MS, ESI-MS/MS, and IR spectra), we have identified that the anti-biofilm molecule is a steroid. In the search for possible mechanisms responsible by anti-biofilm effect of the molecule against P. aeruginosa, 14 genes were evaluated by qRT-PCR. The anti-biofilm effect is not due to a quorum quenching mechanism, as the treated and untreated cells showed similar levels of mRNA for the three major quorum sensing regulators LasR, RhlR and MvfR. Indeed, we show that the molecule inhibits the expression of fliC (flagellin) and cdrA (biofilm scaffold), whose products are necessary for biofilm formation in P. aeruginosa. We found that the compound increases swarming motility. Interestingly, when c-di-GMP levels were increased by overexpression of a diguanilate cyclase, the molecule no longer inhibited biofilm formation. We also demonstrate that it prevents biofilm formation by Staphylococcus epidermidis, in this case by acting as a bactericide. In summary, our novel data indicates that R. microplus protects their eggs against biofilm colonization. This is the first evidence for a mechanism that protects arthropod eggs against biofilm colonization and may be useful for developing new strategies for tick control and biofilm control.