Following cultivation-dependent and -independent techniques, we investigated the microbiota associated withAssociations of insects with bacteria, protozoa, and fungi are complex and intimate, ranging from parasitism to mutualism, and may be extracellular or intracellular and may play a role in the nutrition, the physiology, or the reproduction of the insect host (10). Petri (1909 to 1910) described one of the first bacterial symbiotic associations in an insect species, the olive fly, Bactrocera (Dacus) oleae (31, 32).The olive fruit fly B. oleae is one of the major pests of the olive tree, strongly affecting olive production worldwide, especially in the Mediterranean area, where more than 90% of the world's olive tree cultivation takes place (24,27). Although there have been reports on the isolation of potentially effective Bacillus thuringiensis strains against B. oleae, olive fly control strategies remain almost exclusively based on insecticides, despite the awareness of a need for the use of more environmentally friendly control methods (29). Recently, new concepts are emerging, among which the symbiotic control approach is particularly noteworthy (4). This strategy includes the use of symbionts as vectors of antagonistic factors able to block the life cycle of the plant pathogen in the insect host or, alternatively, their use for the suppression of host natural populations (45). In any case, a prerequisite for developing a symbiotic control approach is the knowledge of the microbiota associated with the insect pest.The nature of the olive fruit fly-associated microbiota is controversial. The culturable bacterium Pseudomonas savastanoi has been suspected to be a mutualist of B. oleae for more than 50 years (6,17,22,25,32,33). In addition, traditional microbiological approaches have identified other bacteria of the genera Bacillus,