**2.1.1 Not only bacteria**

62 Malaria Parasites

response. The effectors of this transmission-blocking immunity are antibody molecules directed against particular protein epitopes (Spano et al., 1996). Anti-Pbs 21 may be used in paratransgenic experiment in the murine system to prove the feasibility of the approach.

Some studies have already shown the possibility to use mosquito guts bacteria to deliver anti-*Plasmodium* effector molecules within the mosquito vector (Riehle and Jacobs-Lorena

In these studies are reported that *E. coli* bacteria expressing SM1 and PLA2, are able to partially inhibit the parasite development. Very recently, Bisi & Lampe (2011) described *Pantoea agglomerans* engineered to express and secrete anti-*Plasmodium* effector proteins. To this purpose, plasmids that included the pelB or hlyA secretion signals from the genes of related species were constructed and tested for their efficacy in secreting SM1, anti-Pbs21,

Very interestingly, *P. agglomerans* was able to secrete HlyA fusions of anti-Pbs21 and PLA2, and now ongoing experiments are evaluating these strains for anti-*Plasmodium* activity in

It is worth to remind that parasites tend to have a heterogeneous genome that favours selection of individuals able to overcome barriers such as drugs or possibly effector gene products favouring the insurgence of resistant strains. Thus the possibility to release paratrasgenic mosquito carrying microbial symbionts modified to express several different

The results cited above, represent a clear "proof of principle" about the applicability of

In this context, the symbiotic bacterium *Asaia* previously described*,* may be very

We engineered *Asaia* strains that were transformed to produce fluorescent proteins; they showed a remarkable capacity to colonize the midgut, reproductive organs ad salivary glands of recipient mosquitoes (Favia et al., 2007; Damiani et al., 2008, 2010). These studies underline the possibility of using this bacterium to express anti-parasite molecules within

Furthermore, *Asaia* can be transmitted, vertically to the progeny (by maternal, paternal and trans-stadial routes) and horizontally between mosquito individuals by mating and co-

As stated above, vertical transmission is important because it offers the chance of introducing engineered bacteria into mosquito populations in the field. In *An. gambiae* it has been proven that vertical transmission occurs by an egg-smearing mechanism, where the extracellular *Asaia* symbionts are smeared onto the egg surface, thus is quite likely that larvae, after emergence, are infected by the bacteria by feeding on them and on egg

Larvae infection by bacteria in the breeding water, offers a way to spread recombinant bacteria in the field, moreover the vertical transmission of *Asaia* symbiont would allow the

molecules would circumvent the selection of resistant malaria strains.

the mosquito body, to inhibit the transmission of the parasite.

feeding (Favia et al., 2007, 2008; Damiani et al., 2008, 2010).

"passage" of recombinant symbionts through the generations.

2005; Riehle et al., 2007).

infected mosquitoes.

successfully implied.

and PLA2 in *P. agglomerans* and *E. coli*.

paratransgenesis in malaria control.

remnants (Damiani et al., 2010).

It is worth to mention that not only bacteria symbiont may find application in paratransgenesis but several evidences indicate that virus, yeast and fungi may also be successfully use.

Ren and collaborators (2008) discovered, cloned and characterized the first known DNV (AgDNV) capable of infection and dissemination in *An. gambiae*. By the development of an AgDNV-based expression vector to express gene(s) of interest in *An. gambiae*, an exogenous gene (enhanced green fluorescent protein; EGFP) was expressed in *An. gambiae* mosquitoes. Wild-type and EGFP-transducing AgDNV virions were able to consistently infect *An. gambiae* larvae, expressed EGFP was detected in adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations.

This work produced clear evidence that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-*Plasmodium* effectors or insect-specific toxins in *Anopheles* mosquitoes.

In the last few months we have identified the yeast *Wickerhamomyces anomalus* as stably associated to some malaria vector species localising at the level of midgut and reproductive organs of the host (Ricci et al., 2011b, 2011c). This localization, the possibility to easily manipulate yeast and the chance to express effector molecules in a eukaryotic organism render *W. anomalus* a potential good candidate for paratransgenesis. Interestingly, very often we found in the midgut and reproductive organs of the mosquito a large number of *Asaia* bacteria in association with a relevant number of *W. anomalus.* This finding may support the idea to use synergistically these two symbionts to release different effector molecules in the mosquito.

We are now proceeding to achieve rapidly a stable transformation system to produce strains of *W. anomalus* able to express and delivery anti-*Plasmodium* peptides.

Very recently, Fang et al (2011) develop transgenic fungi that kill malaria parasite in the mosquito.

They used the fungus *Metarhizium anisopliae* to infect mosquitoes through the cuticle. Recombinant strains of *M. anisopliae* expressing three effector molecules (SM1, a single chain antibody called PfNPNA-1 and an antimicrobial peptide called scorpine) targeting sporozoites during their travel through the hemolymph to the salivary glands were used to infect mosquitoes that had a *Plasmodium*-infected blood meal eleven days before.

The reduction in sporozoite counts was relevant and even more when using *M. anisopliae* strain expressing scorpine and an [SM1](8): scorpine fusion protein (with the reduction in

Facing Malaria Parasite with Mosquito Symbionts 65

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

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

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

The concept that manipulation of microbial symbionts may represent an important tool to

Recent studies have also pointed out that is possible to overcome many of the limitations that since some time ago represented strong limitation to this approach, including the difficulty to culture and transform many symbionts. As discussed in the previous paragraph, to date several symbionts of insect pests and vector borne diseases can be cultivated outside the host, manipulated to express specific factors and reintroduced within the host to produce *in situ* the effector molecules. A very informative example of advanced studies in genetic manipulation of insect symbionts is *Rhodococcus rhodnii*, a bacterial symbiont of the reduviid bug *Rhodnius prolixus.* This bacterium, if manipulated appropriately, can be reintroduced to the vector and inhibit the transmission of *Trypanosoma cruzi* that causes Chagas disease (Beard et al., 2001). Another example regards the bacterium *Alcaligenes*, a gut symbiont of the sharpshooter *Homalodisca coagulata*, the vector of Pierce's

Concerning malaria control, we have described some symbionts that can be cultured outside the mosquito and can be genetically modified to produce specific molecules that have proven to have an inhibitory effect on *Plasmodium* development. In the last 5 years our group has been focused in the study of symbiosis in mosquito vector. Our group as well as others research units, have indicated few microorganisms that possess a strong potential in the paratransgenic control of mosquito-borne diseases and in the very few last years, the number of parasite of medical and veterinary for which the paratransgenic approach has been proposed as one of the element of an integrated control strategy is increasing as demonstrated by study aimed to develop control method of *Leishmania* parasite (Hurwitz et al., 2011). However, even if the genetic modification of insect symbionts to inhibit parasite development, is clearly feasible and achievable in laboratories, many concerns need to be properly addressed before this approach can be applicable in the field. Even though the release of paratransgenic mosquito poses much less ethical and safety concerns that the release of genetically modified mosquitoes, for example the release of large-scale transgenic mosquitoes, would cause not only an increase of the nuisance but also the health risk related to other mosquito borne diseases (it is worth to remind that malaria vector mosquitoes may

contrast insect pests and insect vectors of infectious diseases is now widely accepted.

infection (Walker et al., 2011).

infection.

*An. aegypti* adults (Hoffmann et al., 2011).

**3. Future development and conclusion** 

crop disease (Bextine et al., 2004).

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