RESUMEN
The taxonomic identity of the hereditary prokaryotic symbiont of the olive fly Bactrocera oleae (Diptera: Tephritidae) was investigated. In order to avoid superficial microbial contaminants and loosely associated saprophytic biota, flies were surface-sterilized at the larval stage and reared under aseptic conditions until adult emergence. B. oleae flies originating from different geographical locations and collected at different times of the year were tested. Bacterial isolation was undertaken from the cephalic oesophageal bulb, which is known to be a specific site of accumulation for the hosted microsymbionts in the adult insect. Despite evidence of multiplication cycles taking place within the insect, attempts at cultivation of the isolated bacteria ex situ were not productive at any stage, leading to the choice of unculturable status definition. PCR amplification and nucleotide sequencing of the entire 16S rRNA gene consistently yielded a single sequence that displayed marked similarity with enterobacterial lineages, with closest matches (97%) to Erwinia persicina and Erwinia rhapontici. The novel taxon differs from common intestinal bacterial species of fruit flies and from instances of culturable bacteria previously described in B. oleae raised without sterility precautions, which we also observed as minority occupants or occasional contaminants. The symbiont's identity is also distinct from Pseudomonas savastanoi. In all observations, the numerically dominant inhabitant of the olive fly oesophageal organ was the same unculturable organism, whose presence at later stages was also regularly observed in the midgut. A novel species is proposed, by virtue of its unique properties, under the designation 'Candidatus Erwinia dacicola'.