Symbiotic bacteria inhabiting tephritid flies: a worldwide specific interaction

Several insect lineages have evolved mutualistic associations with their symbiotic bacteria. This is the case of some members of Tephritinae, the most specialized subfamily of fruit flies (Diptera: Tephritidae), harboring co-evolved and vertically transmitted bacterial symbionts in their midgut, known as ‘Candidatus Stammerula spp.’. In the tephritid fly Bactrocera oleae, the major olive pest, symbionts are located in the oesophageal bulb, a diverticulum of the fly head, and designated as ‘Candidatus Erwinia dacicola’. This research, based on two main studies, is focused on different aspects of the relationships between species of the family Tephritidae and their nonculturable symbiotic bacteria. The first study investigated the presence of specific symbiotic bacteria in 15 of the 25 described endemic tephritids of the Hawaiian Archipelago, which represent a spectacular example of adaptive radiation, and their molecular relationships with symbionts of non-Hawaiian tephritids. Moreover the concordant evolution between host and symbiont phylogenies was tested. A specific symbiont was detected through PCR assays in all endemic individuals analyzed and it was designated as ‘Candidatus Stammerula trupaneae’ as it was included in Ca. Stammerula spp. monophyletic clade. The phylogeny of the insect host was reconstructed based on two regions of the mitochondrial DNA (16S rDNA and COI-tRNALeu-COII), while the bacterial 16S rRNA was used for the symbiont analysis. Host and symbiont phylogenies were then compared and evaluated for patterns of cophylogeny and strict cospeciation. Topological congruence between Hawaiian Tephritinae and their symbiotic bacteria phylogenies suggests a limited, but significant degree of host-symbiont cospeciation. The character evolution of three host traits, as island location, host lineage, and host tissue attacked, was finally reconstructed based on the symbiont phylogenies under the hypothesis of cospeciation. The second study surveys the genetic variability of the olive fly symbiont, Ca. Erwinia dacicola, together with the patterns of genetic differentiation of B. oleae, over a large area of its geographical distribution, including most regions of the Mediterranean area, plus South Africa, California and Pakistan. Three bacterial haplotypes, showing a significative geographic distribution, were identified and the co-existence of different Ca. E. dacicola haplotypes in a single fly was never found. Our results reveal the presence of three symbiont haplotypes with a significant phylogeographic distribution related to the territory. In the Mediterranean populations only two bacterial lineages (htA and htB), previously recovered in Italian olive fly populations, have been found, showing a significative East-West genetic differentiation. The South African and Californian olive fly populations were represented only by one of these two lineages, respectively htA and htB. Moreover, a new haplotype (htC) was detected exclusively in the Pakistani population. On the other hand, a high degree of mitochondrial genetic variability with a substantial phylogeographic differentiation has been observed in the B. oleae populations analyzed, revealing the presence of 39 insect haplotypes. Symbiont and host haplotypes were then compared and a significant correlation was found suggesting the predominant presence of vertical transmission. Moreover, the bacterial haplotypes distribution seems to be more related to the territory than the numerous insect host haplotypes, representing an useful tool to reconstruct the debated olive fly’s historical origin