**2. Best symbiont candidates for malaria control**

For malaria Symbiotic Control (SC), "good" candidates are those microbes residing within the mosquito, particularly those localised in the gut. In fact, malarial gametocytes, ingested by a feeding mosquito, must transform to gametes, fuse to form zygotes, and then, as ookinetes, migrate to the mosquito's midgut epithelium to develop as oocysts that release sporozoites to infect the mosquito's salivary glands. At the Oocysts stage of *Plasmodium*, the protozoan parasite causing the malaria infection, represents a favourable target for control method like paratransgenesis aimed to interrupt the disease transmission. In fact, in natural conditions, from thousands of gametocytes ingested by the mosquito vector in an infected blood meal, generally less than ten oocysts will develop.

As stated above, paratransgensis has been already proposed to control insect borne diseases. This is the case of *Rhodnius prolixus*, a triatomine commonly known as the kissing bug, vector of *Trypanosoma cruzi* the causative agent of the Chagas disease. *R. prolixus* harbours in its gut some bacteria that provide nutrients to the insect. These bacteria have been cultivate outside the host, modified to express specific anti-parasite effector molecules and reintroduced within the vector (Beard et al., 2001). These paratransgenic insects have shown to be unable to transmit the parasite. These evidences have posed the basis for the development of paratransgenic-related strategies, based on gut symbionts, to control insect borne diseases.

In the meanwhile they represent the cornerstone on which to base the development of SC of malaria infection.

The search of gut symbiont has been primarily addressed to the identification of bacteria residing in the mosquito gut. Some studies have described the structure of the bacterial community present in several mosquito species (Demaio et al., 1996; Pidiyar et al., 2002,

mosquitoes should be "forced" to host symbionts that have been previously manipulated to express effecto*r* molecules able to interfere with *Plasmodium* development, possibly blocking

In the frame of insect borne diseases, Chagas disease has already proven the feasibility of paratransgenesis in the control of the infection. Vectors of Chagas disease are obligate haematophagous triatomines that feed on vertebrate blood throughout their entire developmental cycle. These insects harbour populations of bacterial symbionts within their intestinal tract, which provide nutrients that insects do not acquire by the blood source. These symbionts have been isolated, cultured outside the host and genetically transformed to express molecules that render the insect refractory to the infection with *Trypanosoma cruzi*, the etiological agent of Chagas disease (Beard et al., 2001). Once reintroduced in the vector

Thus, also on the basis of impulse related to these very exciting results in paratransgenic approach to control Chagas disease, since few years, some researches have been addressed to the identification microbial symbiont that could be implemented in the development of

In this chapter we will give an overview an update of the recent advance in symbiont based-

For malaria Symbiotic Control (SC), "good" candidates are those microbes residing within the mosquito, particularly those localised in the gut. In fact, malarial gametocytes, ingested by a feeding mosquito, must transform to gametes, fuse to form zygotes, and then, as ookinetes, migrate to the mosquito's midgut epithelium to develop as oocysts that release sporozoites to infect the mosquito's salivary glands. At the Oocysts stage of *Plasmodium*, the protozoan parasite causing the malaria infection, represents a favourable target for control method like paratransgenesis aimed to interrupt the disease transmission. In fact, in natural conditions, from thousands of gametocytes ingested by the mosquito vector in an infected

As stated above, paratransgensis has been already proposed to control insect borne diseases. This is the case of *Rhodnius prolixus*, a triatomine commonly known as the kissing bug, vector of *Trypanosoma cruzi* the causative agent of the Chagas disease. *R. prolixus* harbours in its gut some bacteria that provide nutrients to the insect. These bacteria have been cultivate outside the host, modified to express specific anti-parasite effector molecules and reintroduced within the vector (Beard et al., 2001). These paratransgenic insects have shown to be unable to transmit the parasite. These evidences have posed the basis for the development of paratransgenic-related strategies, based on gut symbionts, to control insect

In the meanwhile they represent the cornerstone on which to base the development of SC of

The search of gut symbiont has been primarily addressed to the identification of bacteria residing in the mosquito gut. Some studies have described the structure of the bacterial community present in several mosquito species (Demaio et al., 1996; Pidiyar et al., 2002,

host, the paratransgenic insect vector showed refractoriness to parasite infection.

paratransgenic approach for malaria control.

**2. Best symbiont candidates for malaria control** 

blood meal, generally less than ten oocysts will develop.

transmission.

malaria control.

borne diseases.

malaria infection.

2004; Touré et al., 2000), pinpointing that different mosquito species harbour common bacterial genera (e.g. *Pseudomonas*, *Staphylococcus*, *Enterobacter*, *Escherichia*), some of which might be cultivated outside the vector and manipulated by the means of genetic tools that are already available.

Cultivability in cell-free media and applicability to genetic transformation are two of the fundamental prerequisite of a "good" symbiont to be applied in paratransgenesis control of insect vectors.

More recently, the application of advanced molecular techniques, led to the identification of bacterial species, never described before, has stable component of the microbiota associated to malaria vectors.

For example in the African malaria vector *Anopheles arabiensis* two new species have been recently described (Kämpfer et al., 2006a, 2006b). One was named *Janibacter anophelis* the other *Thorsellia anophelis* to remark the close relationship with the mosquito host. In particular, *T. anophelis* has been proposed in malaria paratransgenic control, since this bacterium, by the means of sequencing of major bands generated in Denaturing Gradient Gel Electrophoresis (DGGE) experiments, appears to be dominant in Kenyan populations of *Anopheles gambiae,* the main African malaria vector (Briones et al., 2008) and dominance is indeed one additional main features required for a symbiont to be employed in paratransgenesis.

The list of bacteria well adapted to malaria vectors midgut has been rapidly growing in the last few years. Among these bacteria two species have been recently described as particularly well adapted to *An. gambiae* midgut, *Pantoea stewartii* and *Elizabethkingia meningoseptica* (Lindh et al., 2008) and thus potentially useful for symbiotic control applications.

Recently, we have identified a Gram-negative -proteobacteria as stably associated to different mosquito species with particular regards to some main malaria vector species (Favia et al., 2007, 2008; Damiani et al., 2008, 2010; Crotti et al., 2009) and its further microbiological and molecular characterization clearly indicate it as belonging to the genus *Asaia* (Ricci et al. 2011a).

The relationship between *Asaia* and mosquito is particularly intriguing in the view of paratransgenic applications since this bacterium localizes in the gut (Figure 1), in the salivary glands and in the reproductive organs of both male and female adult mosquitoes. These localizations are important since in the mosquito gut and salivary glands overlaps with that of *Plasmodium* thus supporting the use of *Asaia* for paratransgenic applications. The localization in the reproductive organs relates to vertical transmission routes that imply favourable means to introduce modified bacteria in the field (Damiani et al., 2010).

Furthermore, *Asaia* is easily cultivable in cell-free media, is detectable at all developmental stage of the mosquito and has high prevalence in mosquito populations. These characteristics have indicated *Asaia* as one of the potentially best bacterial symbiont of malaria vectors to develop paratransgenic protocols to control malaria infection.

Transformation of *Asaia* was originally achieved by a strain originally isolated from *Anopheles stephensi*, one of the major Asian malaria vector, and was attempted by using different plasmid vectors.

Facing Malaria Parasite with Mosquito Symbionts 61

Fig. 3. *Asaia* strain expressing the green fluorescent protein in the midgut of an *An. stephensi*

Some molecules have proven to be effective in inhibiting the development of the malaria parasite within the mosquito vector and may be implemented in paratransgenic

Among them has to be mentioned the small dodecapeptide SM1. This peptide is able to interfere with a binding protein of the lumen of the mosquito midgut and salivary glands that is required for *Plasmodium* invasion. This interference between SM1 and mosquito midgut results in the inhibition of parasite development (Ghosh et al., 2001). A second molecule to be mentioned is the snake venom phospholipase named PLA2 that is able to block *Plasmodium* development within the mosquito by the inhibition of the association

A third molecule is Scorpine an antimicrobial peptide isolated from the venom of the scorpion *Pandinus imperator*, with anti-bacterial activity and a potent inhibitory effect on the ookinete and gamete stages of the rodent malaria parasite *Plasmodium berghei* development

Other effector molecules to be mentioned are two single chain antibodies called anti-

The single-chain antibody PfNPNA-1 is based on a recombinant human monoclonal antibody that specifically recognizes the repeat region (Asn-Pro-Asn-Ala) of the *Plasmodium falciparum* surface circumsporozoite protein and agglutinates sporozoites (Chappel et al.,

Pbs21 is an ookinete surface protein of *P. berghei*, a rodent's parasite, belonging to a class of sexual stage antigens able to induce in the vertebrate host a transmission-blocking immune

**2.1 Effector molecules with anti-***Plasmodium* **effects**

between ookinetes and midgut surface (Zieler et al., 2001).

experiments aimed to block malaria transmission.

male specimen.

(Conde et al., 2000).

2004).

PfNPNA1 and anti-Pbs 21 respectively.

Among these, the plasmid pHM2 was the most efficient and transformed *Asaia* with high efficiency. The gene cassette coding for the green fluorescent protein (Gfp) was cloned into the plasmid vector pHM2 thus providing an efficiently detectable marker to trace mosquito body colonization. Transformed *Asaia* cells were found to efficiently express the protein and showed bright fluorescence useful for localization of the symbiotic cells in the insect body detectable by fluorescent confocal laser scanning microscopy (Figures 2 and 3). Furthermore, in laboratory, when we proposed fed mosquitoes by cotton pad soaked in a mixture of sugar and "green" bacteria, nearly 100% of the recipient populations resulted colonised by the modified *Asaia*.

Fig. 1. *Asaia* bacteria in the gut of an adult *An. stephensi* mosquito.

Fig. 2. *Asaia* strain expressing the green fluorescent protein in the midgut of an *Anopheles stephensi* female specimen.

Among these, the plasmid pHM2 was the most efficient and transformed *Asaia* with high efficiency. The gene cassette coding for the green fluorescent protein (Gfp) was cloned into the plasmid vector pHM2 thus providing an efficiently detectable marker to trace mosquito body colonization. Transformed *Asaia* cells were found to efficiently express the protein and showed bright fluorescence useful for localization of the symbiotic cells in the insect body detectable by fluorescent confocal laser scanning microscopy (Figures 2 and 3). Furthermore, in laboratory, when we proposed fed mosquitoes by cotton pad soaked in a mixture of sugar and "green" bacteria, nearly 100% of the recipient populations resulted colonised by the modified *Asaia*.

Fig. 1. *Asaia* bacteria in the gut of an adult *An. stephensi* mosquito.

*stephensi* female specimen.

Fig. 2. *Asaia* strain expressing the green fluorescent protein in the midgut of an *Anopheles* 

Fig. 3. *Asaia* strain expressing the green fluorescent protein in the midgut of an *An. stephensi* male specimen.

### **2.1 Effector molecules with anti-***Plasmodium* **effects**

Some molecules have proven to be effective in inhibiting the development of the malaria parasite within the mosquito vector and may be implemented in paratransgenic experiments aimed to block malaria transmission.

Among them has to be mentioned the small dodecapeptide SM1. This peptide is able to interfere with a binding protein of the lumen of the mosquito midgut and salivary glands that is required for *Plasmodium* invasion. This interference between SM1 and mosquito midgut results in the inhibition of parasite development (Ghosh et al., 2001). A second molecule to be mentioned is the snake venom phospholipase named PLA2 that is able to block *Plasmodium* development within the mosquito by the inhibition of the association between ookinetes and midgut surface (Zieler et al., 2001).

A third molecule is Scorpine an antimicrobial peptide isolated from the venom of the scorpion *Pandinus imperator*, with anti-bacterial activity and a potent inhibitory effect on the ookinete and gamete stages of the rodent malaria parasite *Plasmodium berghei* development (Conde et al., 2000).

Other effector molecules to be mentioned are two single chain antibodies called anti-PfNPNA1 and anti-Pbs 21 respectively.

The single-chain antibody PfNPNA-1 is based on a recombinant human monoclonal antibody that specifically recognizes the repeat region (Asn-Pro-Asn-Ala) of the *Plasmodium falciparum* surface circumsporozoite protein and agglutinates sporozoites (Chappel et al., 2004).

Pbs21 is an ookinete surface protein of *P. berghei*, a rodent's parasite, belonging to a class of sexual stage antigens able to induce in the vertebrate host a transmission-blocking immune

Facing Malaria Parasite with Mosquito Symbionts 63

Paternal transmission is also important because offers an alternative way to release engineered bacteria in the field in addition to their release in the larval breeding site to contaminate larvae and consequently adults by trans-stadial routes. Indeed, paternal transmission suggests the possibility of releasing not biting male mosquitoes that would be

Ongoing studies are intended to modify *Asaia* to produce strains able to express and secrete anti-*Plasmodium* effector molecules and to test their ability in blocking malaria transmission.

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

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

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 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

We are now proceeding to achieve rapidly a stable transformation system to produce strains

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

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

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

infect mosquitoes that had a *Plasmodium*-infected blood meal eleven days before.

previously colonised with engineered bacteria (Damiani et al., 2010).

ovaries and were transmitted to subsequent mosquito generations.

of *W. anomalus* able to express and delivery anti-*Plasmodium* peptides.

**2.1.1 Not only bacteria** 

in *Anopheles* mosquitoes.

mosquito.

mosquito.

successfully use.

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 2005; Riehle et al., 2007).

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

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 infected mosquitoes.

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 molecules would circumvent the selection of resistant malaria strains.

The results cited above, represent a clear "proof of principle" about the applicability of paratransgenesis in malaria control.

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

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

Furthermore, *Asaia* can be transmitted, vertically to the progeny (by maternal, paternal and trans-stadial routes) and horizontally between mosquito individuals by mating and cofeeding (Favia et al., 2007, 2008; Damiani et al., 2008, 2010).

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 remnants (Damiani et al., 2010).

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 "passage" of recombinant symbionts through the generations.

Paternal transmission is also important because offers an alternative way to release engineered bacteria in the field in addition to their release in the larval breeding site to contaminate larvae and consequently adults by trans-stadial routes. Indeed, paternal transmission suggests the possibility of releasing not biting male mosquitoes that would be previously colonised with engineered bacteria (Damiani et al., 2010).

Ongoing studies are intended to modify *Asaia* to produce strains able to express and secrete anti-*Plasmodium* effector molecules and to test their ability in blocking malaria transmission.
