Environmental Microbiology (2009) 11(12), 3252–3264 doi:10.1111/j.1462-2920.2009.02048.x Asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and ordersemi_2048 3252..3264 Elena Crotti,1§ Claudia Damiani,2§ Massimo Pajoro,3§ malaria mosquito vector Anopheles stephensi, is also Elena Gonella,3§ Aurora Rizzi,1 Irene Ricci,2 present in, and capable of cross-colonizing other Ilaria Negri,3 Patrizia Scuppa,2 Paolo Rossi,2 sugar-feeding insects of phylogenetically distant Patrizia Ballarini,4 Noura Raddadi,1,3† genera and orders. PCR, real-time PCR and in situ Massimo Marzorati,1‡ Luciano Sacchi,5 hybridization experiments showed Asaia in the body Emanuela Clementi,5 Marco Genchi,5 of the mosquito Aedes aegypti and the leafhopper Mauro Mandrioli,6 Claudio Bandi,7 Guido Favia,2 Scaphoideus titanus, vectors of human viruses Alberto Alma3 and Daniele Daffonchio1* and a grapevine phytoplasma respectively. Cross- 1Dipartimento di Scienze e Tecnologie Alimentari e colonization patterns of the body of Ae. aegypti, Microbiologiche, Università degli Studi di Milano, 20133 An. stephensi and S. titanus have been documented Milan, Italy. with Asaia strains isolated from An. stephensi 2Dipartimento di Medicina Sperimentale e Sanità or Ae. aegypti, and labelled with plasmid- or Pubblica, Università degli Studi di Camerino, 62032 chromosome-encoded fluorescent proteins (Gfp and Camerino, Italy. DsRed respectively). Fluorescence and confocal 3Dipartimento di Valorizzazione e Protezione delle microscopy showed that Asaia, administered with the Risorse Agroforestali, Università degli Studi di Torino, sugar meal, efficiently colonized guts, male and female 10095 Turin, Italy. reproductive systems and the salivary glands. The 4Dipartimento di Biologia Molecolare, Cellulare ed ability in cross-colonizing insects of phylogenetically Animale, Università degli Studi di Camerino, 62032 distant orders indicated that Asaia adopts body inva- Camerino, Italy. sion mechanisms independent from host-specific bio- 5Dipartimento di Biologia Animale, Università degli Studi logical characteristics. This versatility is an important di Pavia, 27100 Pavia, Italy. property for the development of symbiont-based 6Dipartimento di Biologia Animale, Università degli Studi control of different vector-borne diseases. di Modena e Reggio Emilia, 41125 Modena, Italy. 7Dipartimento di Patologia Animale, Igiene e Sanità Introduction Pubblica Veterinaria, Università degli Studi di Milano, 20133 Milan, Italy. Microorganisms play crucial roles in the biology and life cycle of most arthropod species, affecting nutrition, devel- opment, reproduction, immunity, defence against natural Summary enemies and speciation (Dale and Moran, 2006; Moran, Bacterial symbionts of insects have been proposed for 2006; Feldhaar and Gross, 2009). Animal symbioses are blocking transmission of vector-borne pathogens. categorized according to the extent of dependence However, in many vector models the ecology of sym- between the host and the symbiont, which generally bionts and their capability of cross-colonizing differ- depends on evolutionary antiquity of the symbiosis. While ent hosts, an important feature in the symbiotic control obligate primary symbionts are essential for the host sur- approach, is poorly known. Here we show that the vival and/or reproduction, secondary symbionts are facul- acetic acid bacterium Asaia, previously found in the tative and thought to be of more recent acquisition, even though they can contribute to the fitness of the host, e.g. conferring resistance to parasites (Dale and Moran, Received 22 December, 2008; accepted 31 July, 2009. *For correspondence. E-mail [email protected]; Tel. (+39) 2006). Most primary symbionts are vertically transmitted 250319117; Fax (+39) 250319238. Present addresses: †Dipartimento to the progeny with a process starting at early stages of di Valorizzazione e Protezione delle Risorse Agroforestali, Università oogenesis or embryogenesis. Vertical transmission is degli Studi di Torino, 10095 Turin, Italy; ‡Laboratory for Microbial Ecology and Technology (LabMET), Ghent University, B9000 Ghent, common also in secondary symbionts, but they can also Belgium. §These authors contributed equally to this work. colonize novel hosts through horizontal transmission © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd Asaia cross-colonization of different insects 3253 among host individuals belonging to the same or different bacterium in other insect vectors (i.e. other Anopheles species (Dale and Moran, 2006). species and S. titanus) raise the question of whether, and Representatives from the group of the acetic acid bac- to which extent, this bacterium can cross-colonize differ- teria (AAB), e.g. Acetobacter, Gluconacetobacter and ent insect hosts. This capacity would allow investigating Gluconobacter, have recently been demonstrated to be the basis of host–symbiont specific interaction and could naturally associated, not only with plants (Kommanee be very promising towards the development of Asaia- et al., 2008), but also with insects, such as the fruit fly based symbiotic control approaches to block parasite Drosophila melanogaster (Corby-Harris et al., 2007; Cox transmission by insect vectors. A high specificity of the and Gilmore, 2007; Ren et al., 2007; Ryu et al., 2008), the host–symbiont interaction characterizes primary sym- olive fly Bactrocera oleae (Kounatidis et al., 2009), the bionts that are vertically transmitted to the progeny but are honeybee Apis mellifera (Jeyaprakash et al., 2003; Mohr not cultivable. This hampers their use for symbiotic and Tebbe, 2006; Babendreier et al., 2007) and the pink control. On the other hand, secondary symbionts include sugarcane mealybug Saccharococcus sacchari (Ashbolt culturable microorganisms that are transmitted both ver- and Inkerman, 1990). Cox and Gilmore (2007) found that tically and horizontally. For symbiotic control applications, in D. melanogaster, Acetobacter was one of the most multiple transmission routes and broad host range carry abundant genera accounting for 29% of all phylotypes. some potential risks regarding biosafety of the biocontrol The detection of AAB in the above insects, all having plant symbiotic microorganism, as well as opportunities for a materials and sugar-rich matrices as food sources, successful disease transmission control. The more the suggests that these bacteria might play roles in food transmission routes, the higher the chances of success exploitation. are. In addition, if a symbiont is present in many different Among AAB, the genus Asaia differentiates because it hosts, the chances of environmental spread and horizon- does not (or weakly) oxidize ethanol to acetic acid. tal transmission to the target species are higher. In this Besides tropical plants, where it was originally isolated work, the capacity of Asaia symbionts to cross-colonize (Yamada et al., 2000; Katsura et al., 2001; Yukphan et al., insects of phylogenetically distant orders, like Diptera and 2004; Malimas et al., 2008), Asaia has thus far been Hemiptera, has been evaluated by using as models found associated with few insect species. These include An. stephensi and Aedes aegypti (Diptera) and S. titanus the hemipteran Scaphoideus titanus, the leafhopper (Hemiptera). We chose these species according to their vector of the phytoplasma causing Flavescence Dorée, a evolutionary distance, one, Ae. aegypty, phylogenetically severe disease of grapevine (Marzorati et al., 2006), and close to An. stephensi, the other, S. titanus, much more three mosquito vectors of malaria, Anopheles stephensi, distant. The presence of Asaia and the ability to cross- Anopheles maculipennis and Anopheles gambiae. Asaia colonize organs and tissues of these insects were has been found stably associated with larvae and adults assessed by in situ hybridization, quantitative real-time of An. stephensi, dominating the microbiota of the mos- PCR and cultivation, and by in vivo studies with Asaia quito (Favia et al., 2007). The distribution of Asaia in the strains labelled with Gfp or DsRed fluorescent proteins body of An. stephensi has been investigated by the use of after isolation from An. stephensi (Favia et al., 2007) and a strain, previously isolated from the mosquito, after Ae. aegypti. genetic modification to express a green fluorescent protein (Gfp). The Gfp-tagged strain efficiently colonized Results the gut, salivary glands and male and female reproductive Detection of Asaia in Ae. aegypti and S. titanus organs. It is noteworthy that Asaia, after assumption with a sugar-based diet by females, was detected in the gut Electron microscopy observations on midgut sections of and then in the salivary glands of the insect, crucial Ae. aegypti and S. titanus individuals (Fig. 1) allowed us organs for the development of the cycle of the malaria to observe bacterial cells resembling Asaia in both parasites Plasmodium spp. (Favia et al., 2007). By using insects. Figure 1A shows a portion of the midgut of Ae. fluorescent strains it was shown that, in An. stephensi, aegypti filled with bacteria, embedded in an extracellular Asaia is vertically transmitted from the mother to offspring matrix, whose morphology is very similar to that already (Favia et al., 2007), but also undergoes paternal transmis- reported for Asaia in An. stephensi (Favia et al., 2007). sion to the progeny, by the way of venereal transfer from Two morphological signatures, i.e. a