**1. Introduction**

56 Malaria Parasites

[56] Boisson B., Jacques J.C., Choumet V., Martin E., Xu J., Vernick K. & Bourgouin C. (2006)

[57] Chaerkady R., Kelkar D.S., Muthusamy B., Kandasamy K., Dwivedi S.B.,

2006), pp. 1935-1942, 0021-9258.

27.

Gene silencing in mosquito salivary glands by RNAi. *FEBS Lett* 580, 8 (Apr 2006), pp. 1988-1992, 0014-5793.Calvo E., Mans B.J., Andersen J.F. & Ribeiro J.M. (2006) Function and evolution of a mosquito salivary protein family. *J Biol Chem* 281, 4 (Jan

Sahasrabuddhe N.A., et al. (2011) A proteogenomic analysis of *Anopheles gambiae* using high-resolution Fourier transform mass spectrometry. Genome Res. 2011 Jul

> 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 particular interest to malaria control.

> 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 malaria control.

> 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,

<sup>\*</sup>Irene Ricci, Patrizia Scuppa, Claudia Damiani, Paolo Rossi, Aida Capone, Chenoa De Freece, Matteo Valzano, Alessia Cappelli, Michela Mosca and Ulisse Ulissi

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.

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 transmission.

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

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 paratransgenic approach for malaria control.

In this chapter we will give an overview an update of the recent advance in symbiont basedmalaria control.
