A promising acetic acid bacterium for paratransgenic control of malaria vectors

Recently, we described an a-proteobacterium of the genus Asaia that is stably associated with different Anopheles species (Favia et al ., 2007) . Asaia was found to massively colonize the midgut and the male reproductive system of adult An. stephensi. Indeed, in situ hybridization and transmission electron microscopy (TEM) revealed that the midgut of adult An. stephensi harbors dense clusters of bacteria that appear extremely uniform at the ultrastructural level and are embedded within a thick slime matrix (Figure 15 .1A) . Slime matrix is typically produced by the Acetobacteraceae, the bacterial family to which Asaia belongs . At higher magnification, the Gram-negative architecture of Asaia is evident, as well as the filamentous appearance of the nucleoid region surrounded by an electron dense cytoplasm (Figure 15 . 1B) . In addition, in situ hybridization and TEM revealed that Asaia also localizes within the male gonoduct, forming large microcolonies In particular, TEM reveals the presence of plugs of bacterial cells within the male deferent duct, presenting the same overall architecture of bacteria in the gut and embedded within a similar extracellular matrix (Figures 15 2A and B) These observations suggest that Asaia might follow a paternal route of transmission to the offspring, as well as a venereal route for horizontal transmission

Asaia thus appears to be a dominant bacterium within the body of An. stephensi, one of the major malaria vectors in Asia . Molecular and microbial analysis also revealed the presence of Asaia in field collected samples of An. maculipennis, a European mosquito vector, and An. gambiae, the main African malaria vector (Favia et al , 2007)

Asaia shows a series of characteristics that make it one of the best available candidates for development of paratransgenesis-based strategies for the control of malaria Indeed, Asaia is characterized by (1) dominance within the mosquito-associated microflora, as revealed by clone prevalence in 16S rRNA gene libraries and quantitative real-time polymerase chain reaction (qRT-PCR); (2) cultivability in cell-free media; (3) transformability with foreign DNA; (4) wide distribution in the larva and adult mosquito body, as shown by TEM and in situ hybridization; (5) colocalization with malaria parasite (in the midgut and salivary glands), and a further localization in the reproductive organs of both sexes

By the use of a bacterial strain modified to express the green fluorescent protein (GFP) and added to sugar or blood meal, we were able to demonstrate the ability of Asaia to quickly colonize the gut (Figure 15 3), salivary glands, and male reproductive organs of mosquitoes Through mating experiments we also demonstrated the high transmission potential of the symbiont from mosquito parents to the progeny, likely through different mechanisms Asaia is also capable of horizontal infection through an oral route during feeding both in preadult and adult stages and through a venereal pattern during mating in adults This capacity of Asaia of being both vertically and horizontally transmitted in mosquitoes and its capacity for surviving and reproducing in the environment highlight

Figure 15.1 TEM micrographs of the midgut of adult females of Anopheles stephensi, showing: (A) the lumen full of Asaia (asterisks = extracellular slime matrix; mw = midgut wall); (B) details of Asaia symbionts, characterized by the presence of an extracellular slime matrix (asterisks), an electron-dense cytoplasm, and a filamentous nucleoid region .

Figure 15.1 TEM micrographs of the midgut of adult females of Anopheles stephensi, showing: (A) the lumen full of Asaia (asterisks = extracellular slime matrix; mw = midgut wall); (B) details of Asaia symbionts, characterized by the presence of an extracellular slime matrix (asterisks), an electron-dense cytoplasm, and a filamentous nucleoid region .

Figure 15.2 TEM micrographs of the adult male deferent of Anopheles stephensi, showing (A) a plug of Asaia symbionts (dw = deferent wall) and (B) details at higher magnification, illustrating the characteristic architecture of Asaia

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Figure 15.3 (Color figure follows p. 238.) The gut of a female Anopheles stephensi mosquito, colonized by transformed bacteria of the genus Asaia that express the green fluorescent protein (GFP) . A massive colonization is located at the midgut level .

the potential of this bacterium for its use in the control of insect pests and insect vector capacities . It is interesting to note that Asaia has also been found in other insects, including hemipteran vectors of plant diseases (Marzorati et al ., 2006; see also Alma et al . in this volume) This bacterium could thus represent a multipurpose weapon for the control of vector borne diseases, both in the medical and veterinary fields, as well as in the area of plant diseases

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