Presence of specific symbiotic bacteria in flies of the subfamily Tephritinae (Diptera Tephritidae) and their phylogenetic relationships: proposal of 'Candidatus Stammerula tephritidis'.

The presence of symbiotic bacteria in flies belonging to the subfamily Tephritinae, which predominantly infest the flower heads of composite flowers (Asteraceae), was investigated. Twenty-five species of flies, collected mainly in northern Italy, were examined. The bacteria adhered to the midgut epithelium in a space external to the peritrophic membrane and therefore not in direct contact with the gut contents. Specific, unique and live, but unculturable bacteria were consistently found in the majority of the fly species and their presence was also shown to be persistent in flies reared under microbiologically controlled conditions and devoid of any residual culturable intestinal bacteria. Sequencing of the small subunit rRNA gene from the novel bacteria indicated that they belonged to the family Enterobacteriaceae. Three main strongly supported clades were delineated by phylogenetic trees, the first of which featured a coherent set of sequences displaying gene sequence similarities lower than 96 % compared with recognized taxa. The second and third clades featured cases with higher gene sequence similarities to culturable bacteria, including Erwinia persicina and Ewingella americana, respectively. Relative rate tests were supportive of a fast genetic evolution for the majority of the bacterial symbionts of the subfamily Tephritinae. In agreement with the interpretation suggested in 1929 after pioneering observations made by H. J. Stammer, a symbiotic relationship between the novel bacteria and the tephritid flies is postulated. The origin of this apparently polyphyletic relationship is discussed and a novel candidate organism is proposed for the first clade under the designation 'Candidatus Stammerula tephritidis'.