File cannot be found.

the world's leading publisher of

Open Access books

Built by scientists, for scientists

Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)

Countries delivered to Contributors from top 500 universities

most cited scientists

154 TOP 1%

International authors and editors

130,000 155M

Downloads

12.2%

Our authors are among the

Open access books available

5,300

Interested in publishing with us?

Contact book.department@intechopen.com

Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com

File cannot be found.

*School of Bioscience & Biotechnology, University of Camerino, Camerino (MC)* 

Microbial symbiosis is an ubiquitous aspect of insect life and plays a fundamental role in the adaptation of insects to the most diverse environments. A very large proportion of insects are supposed to carry bacterial symbionts (Chaves et al., 2009). The variety of the relationships between symbionts and insects are very wide as well as biological function exerted by the symbionts and their localisation within the host. In fact, some are located within host cells while some others are outside. The genetic modification of microbial symbionts has been identified as novel tools to fight insect pests and vectors of infectious diseases. In this frame, in the last years, the use of manipulated symbiont has attracted a lot of attention for the potential application in the control of mosquito-borne diseases, with

Malaria still represents a major health problem with particular impact in the developing countries. Even though several control tools are employed for malaria control, such as bed nets, Anti-malarial drugs, insecticides and other vector control measures, the disease continue to exert a dramatic health burden on the world human populations. Consequently, new effective tools for malaria control are expected. Human malaria is transmitted by some mosquito species belonging to the genus *Anopheles* and methods aimed to control the malaria infection by blocking the transmission from mosquito to humans are particularly attractive.

Recently, quite a lot of attention has been addressed to the so-called Malaria Symbiotic Control (Favia et al., 2008). In fact, more generally, symbiotic microorganisms offer some possibilities for insect pest management strategies and two approaches are particular attractive: The first one implies the disruption of microbial symbionts required by insect pests. The second approach is the manipulation of microorganisms with major impacts on insect traits contributing to their "pest status" for example their capacity to act as vector for diseases like malaria and others. In particular, the paratransgenic approach looks feasible for

Paratransgenesis is the genetic modification of insect symbionts to express anti-pathogen molecules within the vector to block pathogen transmission. In case of malaria infection,

\*Irene Ricci, Patrizia Scuppa, Claudia Damiani, Paolo Rossi, Aida Capone, Chenoa De Freece, Matteo

**1. Introduction** 

malaria control.

www.intechopen.com

particular interest to malaria control.

Valzano, Alessia Cappelli, Michela Mosca and Ulisse Ulissi

**4** 

*Italy* 

**Facing Malaria Parasite** 

Guido Favia et al.\*

**with Mosquito Symbionts** 

File cannot be found.

58 Malaria Parasites

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.

File cannot be found.

Facing Malaria Parasite with Mosquito Symbionts 59

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

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

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

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

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

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

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

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

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

favourable means to introduce modified bacteria in the field (Damiani et al., 2010).

malaria vectors to develop paratransgenic protocols to control malaria infection.

et al., 2008) and thus potentially useful for symbiotic control applications.

are already available.

insect vectors.

to malaria vectors.

paratransgenesis.

*Asaia* (Ricci et al. 2011a).

different plasmid vectors.

File cannot be found.

60 Malaria Parasites

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.

www.intechopen.com

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

File cannot be found.

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).

www.intechopen.com

2004).

PfNPNA1 and anti-Pbs 21 respectively.

File cannot be found.

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).

File cannot be found.

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.

www.intechopen.com

successfully use.

File cannot be found.

64 Malaria Parasites

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).

www.intechopen.com

**2.1.2 Not only paratransgenesis** 

by the release of this natural killer within mosquito organs.

File cannot be found.

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).

File cannot be found.

66 Malaria Parasites

transmit other pathogens), the release into the field of modified symbionts needs to be approached with caution since particularly bacteria may spread very rapidly by horizontal transfer and colonize non target organisms with still unknown consequence. On the other hand is possible to argue that there is no reason to believe that any of the effector molecules identified to date will have any effects on non-target organisms (especially higher organisms) being specifically intended against *Plasmodium*. Still, it is necessary the release of paratransgenic mosquitoes must refer to previous experiences with different Genetically Modified Organisms (GMO) (i.e. GM plants) taking into account indications from different national and international authorities that have already established legal requirements for

If scientific and ethical concerns have to be properly addressed it is also equally important to address concerns of public perception. First of all it is important to underline that no single approach can be successfully *per se* but only an integrated control program that will merge the benefits of different type of approaches would be effective in controlling malaria

It is important to involve residents of the malaria affected countries and their government in official way. In this context it is particularly important to widely release the results of safety tests regarding the use of paratransgenic mosquitoes without covering any possible risk associated to the use of such an approach pinpointing precisely the "real balance" between potential benefits and risks associated to the implementation of the paratransgenic strategy. Christopher Boete (2011) has just published the results of an important study about the use

This study was performed through a questionnaire addressing questions related to the type of research, the location, the nationality and the perception of the public involvement by scientists. The results indicate that even if malaria researchers agree to interact with a nonscientific audience pinpointing that "*they remain quite reluctant to have their research project submitted in a jargon-free version to the evaluation and the prior-agreement by a group of nonspecialists*". The study shows the importance of fostering structures and processes that could lead to an improved involvement of an unspecialized public in the debates linking scientific,

One more aspect that is very important to guarantee success to the paratransgenic approach is the capacity to integrate laboratory and field work bringing together competences from different disciplines and context to produce a variegate and efficient bulk of skills that will

Before any possible field applications, the next step in the assessment of the paratransgenic approach will take advantage by the so-called "semi-field" studies. They can be conducted in mosquito-proof greenhouses that have been termed "malaria spheres" by Knols and collaborators (2002). The green-houses consist in a space-limited ecosystem that recreates an ecological contest with plants, breeding sites etc in which is possible to perform the tests. These test will give important insights about the dynamic of transmission of a due symbiont, in fact releasing subsequent different small numbers of paratransgenic mosquitoes into a malaria sphere containing non-paratransgenic mosquito population it will be possible to determine to the minimum proportion of paratatransgenic insects that need to

of transgenic mosquitoes as a potential approach to interrupt malaria transmission.

the safety of these products (Aguilera et al., 2011).

technological and public health issues in Africa.

www.intechopen.com

be more effective that the simple sum of independent competences.

infection. This is true also for the paratransgenic approach.

File cannot be found.

Facing Malaria Parasite with Mosquito Symbionts 67

be introduced for the symbiont to spread over the whole population. Furthermore it will be possible to acquire information about the capacity of modified symbiont(s) to compete with the natural microbiota. Moreover, all the data acquired by semi-field studies would also provide valuable parameters for modelling experiments to assess the feasibility of

Even though laboratory and field issues have still to be fully overcame, the fast and growing progress recently made in the field of the SC of insect pest and vectors of diseases, induces a relevant optimism that this approach may be applicable for field testing within the next

We wish to thank Luciano Pasqualini for his valuable technical support during the writing of the chapter. Our researches described in this chapter has been supported by the following agencies: agencies Firb-Ideas (grant RBID082MLZ) and Prin 2007 (grant 2007PK2HB7\_002), both from the Italian Ministry of University and Research (MIUR), and by the EU-FP7 Capacities-Infrastructure 2008 (grant 228421) to G.F. P.R. benefited of travel grant from the

Aguilera, J., Gomes, A.R. & Nielsen K.M. (2011). Genetically modified microbial symbiont as

Beard, C.B., Dotsona, P.M., Pennington, S., Eichler, C., Cordon-Rosales, R. & Durvasular, V.

Bextine, B., Lauzon, C., Potter, S., Lampe, D. & Miller, T.A. (2004). Delivery of a genetically

Boete, C. (2011). Scientists and public involvement: a consultation on the relation between

Briones, A.M., Shililu, J., Githure, J., Novak, R. & Raskin, L. (2008). *Thorsellia anophelis* is the

Chaves, S., Neto, M. & Tenreiro, R. (2009). Insect-symbiont systems: from complex

Chappel, J.A., Hollingdale, M.R. & Kang, A.S. (2004). IgG(4) Pf NPNA-1 a human anti-

paratransgenic control strategy. *Curr Microbiol*, (May 2004), pp. 327-331. Bisi, D.C. & Lampe, D.J. (2011). Secretion of anti-*Plasmodium* effector proteins from a natural

arthropod pest controllers: risk assessment through the European legislations.

(2001). Bacterial symbiosis and paratransgenic control of vector-borne Chagas

marked *Alcaligenes* sp. to the glassy-winged sharpshooter for use in a

*Pantoea agglomerans* isolate by using PelB and HlyA secretion signals. *Appl Environ* 

malaria, vector control and transgenic mosquitoes. *Trans R Soc Trop Med Hyg* (in

dominant bacterium in a Kenyan population of adult *Anopheles gambiae* mosquitoes.

relationships to biotechnological applications. *Biotechnol.* (December 2009), pp.

*Plasmodium falciparum* sporozoite monoclonal antibody cloned from a protected individual inhibits parasite invasion of hepatocytes. *Hum Antibodies*, (December

decade thus offering a new weapon to the arsenal against malaria infection.

*Journal of Applied Entomology,* (August 2011), pp. 494-502.

disease. *Int. J. Parasitol*, (May 2001), pp. 621–627.

*Microbiol*, (July 2011), pp. 4669-4675.

ISME J, (November 2007), pp. 74-82.

introducing GM-symbionts under true field conditions.

**4. Acknowledgment** 

COSTAction FA0701.

press).

1753–1765.

www.intechopen.com

2004), pp. 91-96.

**5. References** 

File cannot be found.

68 Malaria Parasites

Conde, R., Zamudio, F.Z., Rodriguez, M.H. & Possani L.D. (2000). Scorpine, an anti-malaria

Crotti, E., Damiani, C., Pajoro, M., Gonella, E., Rizzi, A., Ricci, I., Negri, I., Scuppa, P., Rossi, P.,

Damiani, C., Ricci, I., Crotti, E., Rossi, P., Rizzi, A., Scuppa, P., Esposito, F., Bandi, C.,

Damiani, C., Ricci, I., Crotti, E., Rossi, P., Rizzi, A., Scuppa, P., Capone, A., Ulissi, U., Epis,

Demaio, J., Pumpuni, C.B., Kent, M. & Beier, J.C. (1996). The midgut bacterial flora of wild

Fang, W., Vega-Rodriguez, J., Ghosh, A.K., Jacobs-Lorena, M., Kang, A. & St Leger, R.J.

Favia, G., Ricci, I., Damiani, C., Raddadi, N., Crotti, E., Marzorati, M., Rizzi, A., Urso, R.,

Favia, G., Ricci, I., Marzorati, M., Negri, I., Alma, A., Sacchi, L., Bandi, C., Daffonchio, D.

Ghosh, A.K., Ribolla, P.E & Jacobs-Lorena, M. (2001). Targeting *Plasmodium* Ligands on

Hoffmann, A.A., Montgomery, B.L., Popovici, J., Iturbe-Ormaetxe, I., Johnson, P.H., Muzzi,

Hughes, G.L., Koga, R., Xue P., Fukatsu, T. & Rasgon, J.L. (2011). *Wolbachia* infections are

Hurwitz, I., Hillesland, H., Fieck, A., Das, P. & Durvasula R. (2011). The paratransgenic sand

genera and orders. *Environ. Microbiol*, (September 2009), pp. 3252-3264.

malaria vectors. *Curr. Biol*, (December 2008), pp. 1087-1088.

*Microb Ecol*, (June 2010), pp. 644-654.

*Med. Hyg,* (February 1996), pp. 219-223.

(March 2007), pp. 9047-9051.

New Haven,CT, USA.

pp. 82.

www.intechopen.com

mosquitoes. *Science*, (February 2011), pp. 1074-1077.

*Natl Acad Sci U S A*, (November 2001), pp. 13278-13281.

*gambiae*. *PLoS Pathog*, (May 2011), pp. e1002043.

165-168.

and anti-bacterial agent purified from scorpion venom. *FEBS Lett*, (April 2000), pp.

Ballarini, P., Raddadi N., Marzorati,. M, Sacchi, L., Clementi E., Genchi, M., Mandrioli, M., Bandi, C., Favia,. G, Alma, A. & Daffonchio, D. (2009). *Asaia*, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant

Daffonchio, D. & Favia, G. (2008). Paternal transmission of symbiotic bacteria in

S., Genchi, M., Sagnon, N., Faye, I., Kang, A., Chouaia, B., Whitehorn, C., Moussa, G.W., Mandrioli, M., Esposito, F., Sacchi, L., Bandi, C., Daffonchio, D. & Favia, G. (2010). Mosquito-Bacteria Symbiosis: The Case of *Anopheles gambiae* and *Asaia*.

*Aedes triseriatus*, *Culex pipiens*, and *Psorophora columbiae* mosquitoes. *Am. J. Trop.* 

(2011). Development of transgenic fungi that kill human malaria parasites in

Brusetti, L., Borin, S., Mora, D., Scuppa, P., Pasqualini, L., Clementi, E., Genchi, M., Corona, S., Negri, I., Grandi, G., Alma, A., Kramer, L., Esposito, F., Bandi, C., Sacchi, L. & Daffonchio, D. (2007). Bacteria of the genus *Asaia* stably associate with *Anopheles stephensi*, an Asian malarial mosquito vector. *Proc. Natl. Sci. U S A*,

(2008). Bacteria of the genus *Asaia*: a potential paratransgenic weapon against malaria, In:*Transgenesis and the Management of Vector-Borne Disease*, (Askoy, Serap Ed.), pp. 49-59, ISBN 978-0-387-78224-9, Yale University School of Public Health,

Mosquito Salivary Glands and Midgut with a Phage Display Peptide Library. *Proc*

F., Greenfield, M., Durkan, M., Leong, Y.S., Dong, Y., Cook, H., Axford, J., Callahan, A.G., Kenny, N., Omodei, C., McGraw, E.A., Ryan, P.A., Ritchie, S.A., Turelli, M. & O'Neill, S.L. (2011). Successful establishment of *Wolbachia* in *Aedes* populations to suppress dengue transmission. *Nature*, (August 2011), pp. 454-457.

virulent and inhibit the human malaria parasite *Plasmodium falciparum* in *Anopheles* 

fly: a platform for control of *Leishmania* transmission. *Parasit Vectors*, (May 2011),

File cannot be found.

Facing Malaria Parasite with Mosquito Symbionts 69

Jin, C., Ren, X. & Rasgon, J.L. (2009). The virulent *Wolbachia* strain wMelPop efficiently

Kambris, Z., Blagborough, A.M., Pinto, S.B., Blagrove, M.S., Godfray, H.C., Sinden, R.E. &

Kämpfer, P., Terenius, O., Lindh, J.M. & Faye. I. (2006). *Janibacter anophelis* sp. nov., isolated

Kämpfer, P., Lindh, J.M., Terenius, O., Haghdoost, S., Falsen, E., Busse, H.J. & Faye, I.

Knols, B.G., Njiru, B.N., Mathenge, E.M., Mukabana, W.R., Beier, J.C., Killeen, G.F. (2002)

Lindh, J.M., Borg-Karlson, A.K. & Faye, I. (2008). Transstadial and horizontal transfer of

response to bacteria-containing water. *Acta Trop*, (July 2008), pp. 242-250. Magliani, W., Conti, S., Arseni, S., Frazzi, R., Salati A. & Polonelli L. (2001) Killer anti-idiotypes

Pidiyar, V., Kaznowski, A., Narayan, N.B., Patole, M. & Shouche, Y.S. (2002). *Aeromonas* 

Pidiyar, V.J., Jangid, K., Patole, M.S. & Shouche, Y.S. (2004). Studies on cultured and

Rasgon, J.L. & Scott, T.W. (2004). Phylogenetic characterization of *Wolbachia* symbionts

Ren, X., Hoiczyk, E., Rasgon, J.L. (2008) Viral paratransgenesis in the malaria vector

Ricci, I., Cancrini, G., Gabrielli S., D'Amelio, S. & Favia G. (2002) Searching for *Wolbachia*

Ricci, I., Damiani, C., Rossi, P., Capone, A., Scuppa, P., Cappelli, A., Ulissi, U., Mosca, M.,

Ricci, I., Mosca, M., Valzano, M., Damiani, C., Scuppa, P., Rossi, P., Crotti, E., Cappelli, A.,

*Int. J. Syst. Evol. Microbiol,* (February 2006), pp. 335-338.

*Microbiol,* (May 2009), pp. 3373-3376.

(September 2002), pp. 1723-1728.

*J Med Entomol*, (November 2004), pp. 1175-1178.

*Antonie Van Leeuwenhoek*, (January 2011), pp. 43-50.

*Anopheles gambiae*. *PLoS Pathog.*, (August 2008), e1000135.

(June 2004), pp. 597-603.

(December 2010), pp. 487-493.

www.intechopen.com

e1001143.

pp. 389-392.

establishes somatic infections in the malaria vector *Anopheles gambiae*. *Appl Environ* 

Sinkins S.P. (2010). *Wolbachia* stimulates immune gene expression and inhibits *Plasmodium* development in *Anopheles gambiae*. *PLoS Pathog*, (October 2010), pp.

from the midgut of *Anopheles arabiensis*. *Int. J. Syst. Evol. Microbiol*, (February 2006),

*Thorsellia anophelis* gen. nov., sp. nov., a new member of the Gammaproteobacteria.

MalariaSphere: a greenhouse-enclosed simulation of a natural *Anopheles gambiae* (Diptera: Culicidae) ecosystem in western Kenya. *Malar J*,(December 2002), pp.1-19.

bacteria within a colony of *Anopheles gambiae* (Diptera: Culicidae) and oviposition

in the control of fungal infections. *Curr Opin Investig Drugs*, (April 2001), pp. 477-479.

*culicicola* sp. nov., from the midgut of *Culex quinquefasciatus*. *Int J Syst Evol Microbiol*,

uncultured microbiota of wild *Culex quinquefasciatus* mosquito midgut based on 16s ribosomal RNA gene analysis. *The American Journal of Tropical Medicine and Hygiene,* 

infecting *Cimex lectularius* L. and *Oeciacus vicarius* Horvath (Hemiptera: Cimicidae).

(Rickettsiales: Rickettsiaceae) in mosquitoes (Diptera: Culicidae): large polymerase chain reaction survey and new identifications. *J Med Entomol,* (July 2002), pp. 562-567.

Valzano M., Epis, S., Crotti, E., Daffonchio, D., Alma, A., Sacchi L., Mandrioli M., Bandi C. & Favia, G. (2011). Mosquito symbioses: from basic research to the paratransgenic control of mosquito-borne diseases. *Journal of Applied Entomology*,

Ulissi, U., Capone, A., Esposito, F., Alma, A., Mandrioli, M., Sacchi, L., Bandi, C., Daffonchio, D. & Favia G. (2011). Different mosquito species host *Wickerhamomyces anomalus* (*Pichia anomala*): perspectives on vector-borne diseases symbiotic control.

File cannot be found.

70 Malaria Parasites

Ricci, I., Damiani, C., Scuppa, P., Mosca, M., Crotti, E., Rossi, P., Rizzi, A., Capone, A.

Riehle, M.A. & Jacobs-Lorena, M. (2005). Using bacteria to express and display anti-parasite

Riehle, M.A., Moreira, C.K., Lampe. D., Lauzon. C. & Jacobs-Lorena, M. (2007). Using

Savoia, D., Avanzini C., Conti, S., Magliani, V., Frazzi R. & Polonelli, L. (2002). In vitro

Séguy, N., Polonelli, L., Dei-Cas, E. & Cailliez J.C. (1998). Effect of a killer toxin of *Pichia* 

Serbus, L.R., Casper-Lindley, C., Landmann F. & Sullivan, W. (2008) The genetics and cell

Sinkins, S.P. (2004). *Wolbachia* and cytoplasmic incompatibility in mosquitoes. *Insect Biochem* 

Spano, F., Matsuoka, H., Ozawa, R., Chinzei, Y. & Sinden R.E. (1996). Epitope mapping on

Tiawsirisup, S., Sripatranusorn, S., Oraveerakul. K. & Nuchprayoon S. (2008). Distribution of

Touré, A.M., Mackey, A.J., Wang, Z.X. & Beier, J.C., (2000). Bactericidal effects of sugar-fed

Walker, T., Johnson, P.H., Moreira, L.A., Iturbe-Ormaetxe, I., Frentiu, F.D., McMeniman,

invades caged *Aedes aegypti* populations. *Nature*, (August 2011), pp. 450-453. Zieler, H., Keister, D.B., Dvorak, J.A. & Ribeiro J.M.(2001) A Snake Venom Phospholipase

(Diptera: Culicidae). *J. Med. Entomol,* (March 2000), pp. 246-249.

2011), pp. 911-21.

2005), pp. 699-707.

707.

4157-4167.

www.intechopen.com

*Int. J. Parasitol,* (May 2007), pp. 595-603.

*Mol Biol,* (July 2004), pp. 723-729.

(December 1996), pp. 559-563.

*Eukaryot Microbiol*, (July 2002), pp. 319-323.

*Immunol Med Microbiol*, (September 1998), pp.145-149.

Thailand. *Parasitol Res*, (March 2008), pp. 731-735.

Gonella, E., Ballarini, P., Chouaia, B., Sagnon, N., Esposito, F., Alma, A., Mandrioli, M., Sacchi, L., Bandi, C., Daffonchio, D. & Favia, G. (2011). The Yeast *Wickerhamomyces Anomalus* (*Pichia Anomala*) Inhabits the Midgut and Reproductive System of the Asian Malaria Vector *Anopheles Stephensi*. *Environ Microbiol,* (April

molecules in mosquitoes: current and future strategies. *Insect Biochem Mol Biol*, (July

bacteria to express and display anti-*Plasmodium* molecules in the mosquito midgut.

leishmanicidal activity of a monoclonal antibody mimicking a yeast killer toxin. *J* 

*anomala* to *Pneumocystis*. Perspectives in the control of pneumocystosis. *FEMS* 

biology of *Wolbachia*-host interactions. *Annu Rev Genet*, (December 2008), pp. 683-

the ookinete surface antigen Pbs21 of *Plasmodium berghei*: identification of the site of binding of transmission-blocking monoclonal antibody 13.1. *Parassitologia*,

mosquito (Diptera: Culicidae) species and *Wolbachia* (Rickettsiales: Rickettsiaceae) infections during the bird immigration season in Pathumthani province, central

antibiotics on resident midgut bacteria of newly emerged anopheline mosquitoes

C.J., Leong, Y.S., Dong, Y., Axford, J., Kriesner P., Lloyd, A.L., Ritchie, S.A., O'Neill, S.L. & Hoffmann A.A. (2011). The wMel *Wolbachia* strain blocks dengue and

a(2) Blocks Malaria Parasite Development in the Mosquito Midgut by Inhibiting Ookinete Association with the Midgut Surface. *J Exp Biol,* (December 2001), pp.

File cannot be found.

Edited by Dr. Omolade Okwa

ISBN 978-953-51-0326-4 Hard cover, 350 pages

**InTech China**

Guido Favia, Irene Ricci, Patrizia Scuppa, Claudia Damiani, Paolo Rossi, Aida Capone, Chenoa De Freece, Matteo Valzano, Alessia Cappelli, Michela Mosca and Ulisse Ulissi (2012). Facing Malaria Parasite with

Mosquito Symbionts, Malaria Parasites, Dr. Omolade Okwa (Ed.), ISBN: 978-953-51-0326-4, InTech, Available from: http://www.intechopen.com/books/malaria-parasites/facing-malaria-parasite-with-mosquito-symbionts-

> Phone: +86-21-62489820 Fax: +86-21-62489821

Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China

**Published online** 30, March, 2012

Malaria is a global disease in the world today but most common in the poorest countries of the world, with 90% of deaths occurring in sub-Saharan Africa. This book provides information on global efforts made by scientist which cuts across the continents of the world. Concerted efforts such as symbiont based malaria control; new applications in avian malaria studies; development of humanized mice to study P.falciparium (the most virulent species of malaria parasite); and current issues in laboratory diagnosis will support the prompt treatment of malaria. Research is ultimately gaining more grounds in the quest to provide vaccine for the prevention of malaria. The book features research aimed to bring a lasting solution to the malaria problem and what we should be doing now to face malaria, which is definitely useful for health policies in the twenty first century.

**Published in print edition** March, 2012

**Publisher** InTech

In order to correctly reference this scholarly work, feel free to copy and paste the following:

**InTech Europe**

**How to reference**

University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com

**Malaria Parasites**

File cannot be found.

© 2012 The Author(s). Licensee IntechOpen. This is an open access article

License, which permits unrestricted use, distribution, and reproduction in

distributed under the terms of the Creative Commons Attribution 3.0

any medium, provided the original work is properly cited.