e. the reappearance of the Asaia bands. In YM155 in vivo summary, our experiments provide evidence that Asaia plays a beneficial function for the normal mosquito larval development. The fact that Asaia is the major inhabitant of the gut in An. stephensi [7], and that it is transmitted EVP4593 mw to the progeny by different ways [7][9], is also in agreement with

the idea that this alpha-proteobacterium has a beneficial role for the insect. Even though we did not generate experimental evidence that could indicate the specific function for Asaia, some hypothesis can be proposed. The negative effects of Asaia loss on the larval growth of An. stephensi increase with the advancement of the development, in parallel with the increased metabolic requirement. We could thus suggest that Asaia is involved in the supply of nutrients to the host, like a nitrogen source [13], or vitamins, or other essential nutritional factors. But this does not exclude the possibility that Asaia can play a role in the development/homeostasis of the immune system of the host, as shown for other acetic acid bacteria that contribute to the proper functioning of the host insect immunity [11]. Conclusions Antibiotic removal of bacterial symbionts is a classic experimental strategy in studies on invertebrate

symbioses. PRI-724 in vivo After administration of an antibiotic to the host, which is supposed to be effective on a given symbiont, physiological/pathological effects on the host are recorded, with the goal of getting clues on the biological role of the symbiont under study [15]. This strategy is however flawed by the multiple effects associated with antibiotic treatments, from direct effects on the host, to effects on other components of the microbiota. Here we have adopted a novel strategy, consisting in the administration antibiotic-resistant symbionts to antibiotic-treated individuals. In our study, the simple observation of a delay in PtdIns(3,4)P2 the development in An. stephensi larvae after rifampicin treatment, in parallel with a dramatic reduction of Asaia burden, led to the hypothesis that this bacterium

plays a beneficial role in the development of the mosquitoes. The restoration of the normal developmental time after administration of rifampicin-resistant Asaia provides a strong support to the above hypothesis. However, our work does not prove that Asaia is necessary for mosquito development. Indeed, we cannot exclude that a normal developmental time could be restored after administration of other microorganisms. On the other side, it is clear that introduction of antibiotic-resistant Asaia is sufficient for restoring mosquito development. In summary, while our results indicate that Asaia is sufficient for allowing a normal mosquito development, we cannot conclude that this bacterium is necessary, since we have not tested the administration of other bacteria.