Phylogenetic studies of tephritid flies (Diptera, Tephritidae) and their symbiotic bacteria

Tephritidae, commonly known as "fruit flies" is a large and complex family. Most, particularly the frugivorous species, are notorious pests. These include Ceratitis capitata (Wiedemann) (Mediterranean fruit fly), Bactrocera oleae (Rossi) (olive fly), Rhagoletis cerasi (L.) (cherry fly), Rhagoletis completa (Cresson) (walnut husk fly) and other exotic species. Other species, however, live on the flower heads of Asteraceae. Since the beginning of the last century, some authors (Petri 1909, Stammer, 1929) report the presence of symbiotic bacteria in flies belonging to the subfamily Tephritinae. Recently the olive fly symbiont has been described and designated as 'Candidatus Erwinia dacicola' by (Capuzzo et al., 2005). The present work aims to study the relationships between species of the family Tephritidae and their symbiotic bacteria. It is based upon three main studies, the first of which has already been published. 1.- The first study is the continuation of Alessia Piscedda PhD. thesis and deals with the identity of symbiotic bacteria, in 25 flies belonging to the subfamily Tephritinae (Diptera: Tephritidae), which were collected mainly in northern Italy. In order to detect and identify symbiotic bacteria, the first tract of the midgut of flies emerging from previously sterilized pupae, was plated on different microbiological media, LIVE/DAD BacLight staining was performed and biomolecular techniques were used. According to Stammer, (1929) the presence of non culturable symbiotic bacteria has been detected in species of genera Tephritis, Campiglossa, Trupanea, Acanthiophilus, Sphenella, and Oxyna. Symbiotic bacteria have also been found in other genera (Capitites, Dioxyna, Noeeta), which were not studied by Stammer. Sequencing 1000 bp of the small subunit rDNA gene from these symbiotic bacteria has indicated that they belong to the family Enterobacteriaceae and a novel candidate organism has been proposed for the symbiotic bacteria of the genus Tephritis, under the designation 'Candidatus Stammerula tephritidis'. These analyses have been extended to other tribes of the subfamily Tephritinae (Xyphosiini, Myopitini e Terellini), using the same techniques reported above, but non symbiotic bacteria have been detected in these tribes, as suggested by Stammer (1929). 2.- The second study of the present work analyzes the phylogenetic relationships between tephritid flies of the subfamily Tephritinae. Two regions of the mitochondrial DNA, 16S rDNA e COI-tRNALeu-COII, were examined. The phylogenetic trees obtained from a Bayesian Inference and a Maximum-Likelihood analysis have suggested, as a rule, the presence of five monophyletic clusters corresponding to the fives tribes of this subfamily: Tephritini, Myopitini, Xyphosiini, Noeetini e Terellini. The phylogenetic tree obtained from the analysis of the COI-tRNALeu-COII showed more highly resolved trees and the internal nodes more highly supported than the phylogeny inferred from the 16S data set, and defined the relationships among the tribes better. Cophylogenetic analysis has been carried out, and the presence of congruence between hosts and symbionts, even if imperfect, has been suggested. The reconstructions obtained showed two principal events. The most important and probably earliest event corresponds with the acquisition of symbiotic bacteria by the common ancestor of the tribe. The presence of non-strict congruence is probably due to other events such as losses, duplications and hostswitchings. Indeed, these bacteria are extracellular symbionts and some opportunities for host-switching occur during the biological cycle of the fly. In the larval stadium, for instance, bacteria are located in the intestinal caeca (Petri 1909; Stammer, 1929), without the protection of the peritrophic membrane and are thus, in contact with free living bacteria present in the intestinal lumen. The contemporaneous presence of different species in the same host plant could also be an opportunity for host-switching. Considering all of these aspects, the presence of congruence, even if not strict, results particularly interesting and a physiological compatibility between host and symbiont seems to appear. 3.- In the third part of my PhD. thesis, the phylogenetic analysis of insects has been extended to Paleartic species belonging to other subfamilies (Trypetinae e Dacinae). It has been based on the analysis of two regions of the mitochondrial DNA: 16S e COI-tRNALeu-COII. The availability of sequences of the 16S rDNA of several species in GenBank, has allowed extending this data set. These phylogenetic analysis still in progress, confirms the traditional classification based on a morphological approach but suggests also interesting relationships among the tribes. I have also attempted to associate the phylogeny obtained with morphological symbiotic arrangements and biological characteristics. Interestingly, it was pointed out that all the species of the subfamily Tephritinae that overwinter as adults, present symbiotic bacteria in the first tract of the midgut. The presence of these bacteria seems to be essential for the overwintering adults. Indeed, while the diet of larval stages includes relatively rich substrates such as flower tissue and seeds, glyciphagous adults have access to less resources. Thus the presence of bacteria could be more critical for their survival than that in the earlier stages.