**2.1.2 Not only paratransgenesis**

Symbiont may be successfully implied in SC of mosquito vector even without their genetic manipulation. This would be the case of *W. anomalus*. In fact several strains of this yeast produce killer toxins with an antimicrobial effect on a wide spectrum of human pathogens (Séguy et al., 1998; Magliani et al., 2001), including insect-transmitted protozoan parasites such as *Leishmania* spp. (Savoia et al., 2002). Preliminary data seems to confirm that the *W. anomalus* strain isolated from *An. stephensi*, produces a killer toxin that is active against some yeast strains (Ricci et al. manuscript in preparation). We will soon test if this killer toxin is active against *Plasmodium* to verify the possibility to contrast malaria parasite development by the release of this natural killer within mosquito organs.

One mosquito symbiont that has been extensively studied is the alpha-proteobacterium *Wolbachia*, a very common cytoplasmic symbiont of the majority of insect species, crustaceans, mites, and filarial nematodes (Serbus et al., 2008). *Wolbachia* are maternally inherited bacteria that inducing cytoplasmic incompatibility in mosquitoes that they use to spread themselves through populations enhancing their transmission (Sinkins, 2004). This ability of self-spreading through mosquito populations attracted a lot of attention in the last few years and has been proposed as a gene drive system for mosquito genetic replacement, for the reduction of population size or for modulating population age structure to reduce disease transmission.

Interestingly, even though the very wide range of insect species naturally infected by *Wolbachia*, natural infection has never been detected in species belonging to the genus *Anopheles* (Ricci et al., 2002; Rasgon & Scott, 2004; Tiawsirisup et al., 2008). On the other hand cultured *Anopheles* cells can be infected (Jin et al., 2009), this has opened new possibility to study perspectives in the use of endosymbiont to control malaria infection.

An "over-replicating" strain of *Wolbachia pipientis* named wMelPop has recently been shown to induce immune upregulation and inhibition of pathogen transmission in *Aedes aegypti* the principal mosquito vector of dengue viruses, thus transient somatic infections of wMelPop were performed by intrathoracic inoculation in *An. gambiae*. As consequence of these inoculations, up-regulation of several specific immune genes was detected and some of these have shown a direct influence on the development of malaria parasites. Similar results have been achieved when using a stably infected *An. gambiae* cell line (Kambris et al., 2010).

Other studies have characterized somatic infections of two diverse *Wolbachia* strains (wMelPop and wAlbB) in *An. gambiae*. After infection, wMelPop was localized in fat body, head, sensory organs and other tissues but was not detected in midgut and ovaries, even though both *Wolbachia* strains inhibit *P. falciparum* oocyst in the mosquito midgut.

Even more interestingly, although not virulent in non-bloodfed mosquitoes, wMelPop was found to be virulent for around 12-24 hours post-bloodmeal. All these data strongly indicate that if stable transinfections would behave similarly to somatic infections, *Wolbachia* could potentially be used as part of a strategy to control malaria-transmitting mosquitoes (Hughes et al., 2010).

sporozoite counts of 98%). These experiments prove that *Metarhizium*-mediated inhibition of *Plasmodium* development could be an additional tool to contrast malaria development

Symbiont may be successfully implied in SC of mosquito vector even without their genetic manipulation. This would be the case of *W. anomalus*. In fact several strains of this yeast produce killer toxins with an antimicrobial effect on a wide spectrum of human pathogens (Séguy et al., 1998; Magliani et al., 2001), including insect-transmitted protozoan parasites such as *Leishmania* spp. (Savoia et al., 2002). Preliminary data seems to confirm that the *W. anomalus* strain isolated from *An. stephensi*, produces a killer toxin that is active against some yeast strains (Ricci et al. manuscript in preparation). We will soon test if this killer toxin is active against *Plasmodium* to verify the possibility to contrast malaria parasite development

One mosquito symbiont that has been extensively studied is the alpha-proteobacterium *Wolbachia*, a very common cytoplasmic symbiont of the majority of insect species, crustaceans, mites, and filarial nematodes (Serbus et al., 2008). *Wolbachia* are maternally inherited bacteria that inducing cytoplasmic incompatibility in mosquitoes that they use to spread themselves through populations enhancing their transmission (Sinkins, 2004). This ability of self-spreading through mosquito populations attracted a lot of attention in the last few years and has been proposed as a gene drive system for mosquito genetic replacement, for the reduction of population size or for modulating population age structure to reduce

Interestingly, even though the very wide range of insect species naturally infected by *Wolbachia*, natural infection has never been detected in species belonging to the genus *Anopheles* (Ricci et al., 2002; Rasgon & Scott, 2004; Tiawsirisup et al., 2008). On the other hand cultured *Anopheles* cells can be infected (Jin et al., 2009), this has opened new possibility to study perspectives in the use of endosymbiont to control malaria infection.

An "over-replicating" strain of *Wolbachia pipientis* named wMelPop has recently been shown to induce immune upregulation and inhibition of pathogen transmission in *Aedes aegypti* the principal mosquito vector of dengue viruses, thus transient somatic infections of wMelPop were performed by intrathoracic inoculation in *An. gambiae*. As consequence of these inoculations, up-regulation of several specific immune genes was detected and some of these have shown a direct influence on the development of malaria parasites. Similar results have

Other studies have characterized somatic infections of two diverse *Wolbachia* strains (wMelPop and wAlbB) in *An. gambiae*. After infection, wMelPop was localized in fat body, head, sensory organs and other tissues but was not detected in midgut and ovaries, even

Even more interestingly, although not virulent in non-bloodfed mosquitoes, wMelPop was found to be virulent for around 12-24 hours post-bloodmeal. All these data strongly indicate that if stable transinfections would behave similarly to somatic infections, *Wolbachia* could potentially be used as part of a strategy to control malaria-transmitting mosquitoes (Hughes

been achieved when using a stably infected *An. gambiae* cell line (Kambris et al., 2010).

though both *Wolbachia* strains inhibit *P. falciparum* oocyst in the mosquito midgut.

within the vector.

disease transmission.

et al., 2010).

**2.1.2 Not only paratransgenesis** 

by the release of this natural killer within mosquito organs.

From this point of view, the "*Ae. aegypti* lesson", can be particularly important. Similar to *Anopheles*, *Ae. aegypti* has never been detected as naturally infected by *Wolbachia*, transinfection of selected strains of *Wolbachia* in *Ae. aegypti* can block the development of dengue infection (Walker et al., 2011).

Very recently the wMel *Wolbachia* strain was introduced into *Ae. aegypti* from *Drosophila melanogaster* showing a successfully invasion two natural *Ae. aegypti* populations in Australia, reaching almost the fixation in a few months after the releases of wMel-infected *An. aegypti* adults (Hoffmann et al., 2011).

These findings demonstrate that *Wolbachia*-based strategies can be a successful approach to eliminate dengue infection and a similar approach may be developed to control malaria infection.
