**3. Future development and conclusion**

The concept that manipulation of microbial symbionts may represent an important tool to contrast insect pests and insect vectors of infectious diseases is now widely accepted.

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 crop disease (Bextine et al., 2004).

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

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.

2004), pp. 91-96.

**5. References** 

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

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 infection. This is true also for the paratransgenic approach.

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

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, technological and public health issues in Africa.

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 be more effective that the simple sum of independent competences.

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 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 introducing GM-symbionts under true field conditions.

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

## **4. Acknowledgment**

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.

be more effective that the simple sum of independent competences.

infection. This is true also for the paratransgenic approach.

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 COSTAction FA0701.

#### **5. References**


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.

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.


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., 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,

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

genera and orders. *Environ. Microbiol*, (September 2009), pp. 3252-3264. Damiani, C., Ricci, I., Crotti, E., Rossi, P., Rizzi, A., Scuppa, P., Esposito, F., Bandi, C.,

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.

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

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

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


**Part 3** 

**Malaria Parasite Research** 

