16S rRNA Gene-Based Identification of Midgut Bacteria from Field-Caught Anopheles gambiae Sensu Lato and A. funestus Mosquitoes Reveals New Species Related to Known Insect Symbionts

Abstract

Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the ␥-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.Malaria remains the parasitic disease that kills the most people in the world. Anopheles gambiae sensu lato and Anopheles funestus mosquitoes are the main vectors in Africa, where 90% of malaria-related deaths occur. An approach to stop malaria transmission is paratransgenics. In this approach, suitable symbiotic bacteria are genetically modified to produce an antiparasitic factor and then reintroduced into the insect gut, where they kill or inhibit the development of the parasites (4).A few studies have been performed to investigate bacterial species in field-collected Anopheles mosquitoes, all using culturing techniques. Jadin et al. (22) To identify bacterial candidates for a paratransgenic mosquito, we conducted a screen for uncultured and cultured midgut bacteria from wild-caught A. gambiae and A. funestus mosquitoes. MATERIALS AND METHODSField site, mosquitoes, and dissections. Indoor-resting, blood-fed female A. gambiae sensu lato and A. funestus mosquitoes were caught in Lwanda, 12 km east of Mbita Point Research and Training Centre, ICIPE, Suba district, Western Kenya. In total, 116 Anopheles mosquitoes were caught on eight different occasions (A2 to H2). Living mosquitoes were anesthetized with chloroform, the species were determined by morphology and PCR (A. gambiae sensu lato) (30a). The mosquitoes were dissected in a sterile hood. Individual midguts were mashed in 50 l of sterile saline (0.9% NaCl); this suspension was later used for isolation of bacteria and cloning of the 16S rRNA gene from bacteria. Controls for the efficiency of sterilization were treated like the other samples.Bacterial isolation and phenotypic characterization. The midgut suspension was streaked on Luria-Bertani agar (LA) plates and incubated for 48 h at room temperature. All bacteria were restreaked and pre...

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