The goal of these investigations was to attempt to observe the horizontal transfer of the endosymbiont Ca. E. dacicola from a wild B. oleae population to a laboratory colony. A secondary goal was to determine the best and most efficient method to reliably screen for this endosymbiont in B. oleae samples. It was predicted that horizontal transfer could occur via both oral contamination (wild flies’ regurgitation on gelled water and olives) and via anus-genital contamination (eggs laid inside oviposition domes by wild flies, wild faeces, and cohabitation with wild flies).
Concerning the oral contamination transmission route and more specifically regurgitation, we tested the hypothesis that flies could regurgitate saliva with bacteria on two different substrates, olives and gelled water. Petri first described this behaviour in 1907 , and he reported a peculiar behaviour of B. oleae in which the fly sucked and regurgitated olive juice during the oviposition process, commonly known as “the kiss” . Tzanakakis  also described this action in B. oleae, assuming that, at the end of the oviposition process, the female retracts the ovipositor and regurgitates the juice sucked from the hole to deter subsequent oviposition. Drew and Lloyd  also described strict relationships between tropical Dacinae and the bacteria of host plants. They showed that the bacteria present in the alimentary tract of flies were also found on the surface of host fruit from plants in which flies had been collected, suggesting that regurgitation was involved in this bacterial presence. However, in our experiment, even if the substrates had been contaminated through bacterial regurgitation by the wild olive fruit fly, the transfer of Ca. E. dacicola to lab flies did not occur, either through the olives or the gelled water. However, no attempts to detect Ca. E. dacicola on these two substrates were carried out, since the transfer did not occur we presume that the symbiont was not present on them or, if present, it was probably not available for the horizontal transfer.
Regarding the possible anus-genital transfer, wax domes containing eggs laid by wild females were tested as a contamination source. The presence of Ca. E. dacicola was found on the eggs, not only by biological molecular techniques  but also by morphological observations dealing with the presence of bacterial colonies around the ano-genital opening and in the micropylar area . Furthermore, previous observations had highlighted the presence of bacterial masses on B. oleae eggs . Since several previous studies demonstrated that Ca. E. dacicola is vertically transmitted from the female to the egg [9, 10, 15, 17, 25]; we predicted that a horizontal transfer mechanism could occur after the lab flies have direct contact with the eggs laid by wild females. However, our attempt was not successful. In terms of vertical transmission, there are many ways to “pass” symbiotically useful bacteria via the egg, from the mother to the progeny. For instance, symbiotic bacteria can be maternally transmitted by “capsule transmission” or by “egg smearing,” as observed in stinkbugs . It could also be transferred to the egg as it passes through the micropyles, as is believed to occur in fruit flies . For the vertical transfer of Ca. E. dacicola in B. oleae, the bacterium seems to be maternally transmitted by “egg smearing” . Thus, even if the endosymbiont is smeared on the egg’s surface, its passage to the young larva is probably strictly related to the micro environment inside the olive. Given these assumptions, we predict that in the present work, this horizontal transfer via egg using wax domes did not occur, perhaps because Ca. E. dacicola on the egg surfaces was exposed to air for too long, instead of remaining in the “small oblong chamber” inside the olive  with low oxygen levels, thus limiting the possibility of horizontal transfer. Another hypothesis could be that after oviposition inside the fruit, the endosymbiont needs some olive compounds that enable it to stay viable until larval assumption.
Because the symbiont passes through and colonizes the digestive tract during the entire adult lifespan , and especially given its role in nitrogen metabolism , we tested the hypothesis that it could be partially released in the faeces after digestion. The endosymbiont was indeed detected on faeces and on sponges taken from the replicates of the faeces treatment. These sponges stayed in contact with the wild flies for a long time (they were inserted during the contamination phase along with wild adults, and they were not exchanged with new sterile sponges for the acquisition phase, as in other theses). We therefore believe that they were contaminated by faeces. However, no horizontal transfer was observed after using this substrate as a contamination source. Based on this, we presume that even if Ca. E. dacicola DNA was detected both on the faeces and sponges, the bacterium may not be viable on these substrates and may not be horizontally transferred in this way. These findings further suggest that Ca. E. dacicola may be a bacterium that needs low levels of oxygen to maintain its vitality and grow.
Consistent with our hypotheses and the results of Estes et al. , horizontal transfer via cohabitation with wild flies was the only treatment in which transfer occurred. To our knowledge, the transmission of Ca. E. dacicola could have occurred through different methods, including mating, coprophagy or trophallaxis. Copulation between males and females was not directly verified; there is a high probability that the flies did mate, but we cannot be sure that this was the way through which the transfer occurred. Further trials assessing cohabitation between wildM x labM or wildF x labF could be set out in order to better clarify this finding. The flies in the cohabitation scenario also had ample opportunities to regurgitate and defecate in the same cage. This observation allowed us to make a second hypothesis: perhaps not only the mating, but also the coprophagy and/or the trophallaxis behaviour between wild and lab flies during their cohabitation accounted for the horizontal transfer. The only thing we know is that the wild and lab flies stayed together for 15 days and they had time to perform other behaviours and to be in contact frequently in different ways. Trophallaxis represents an “exchange of alimentary liquid among colony members and guest organisms,” and it can occur before, during, or after mating. It can also be direct or indirect, stomodeal or proctodaeal, and it has been described in approximately 20 species of Tephritidae, representing a behaviour that involves the transfer of substances . Several studies described the mating trophallaxis in Tephritidae [40,41,42] but did not demonstrate the transfer of any substance during the contact between the mouthparts of the mates. Our results lead us to suppose that this behaviour could be involved in endosymbiont transfer, as predicted by Estes et al. . They hypothesized that bacterial transfer occurs through coprophagy, presumably thanks to pre/in direct proctodaeal trophallaxis. Moreover, it must be noted that we found Ca. E. dacicola DNA inside the oesophageal bulb of lab flies that cohabited with wild flies; as a consequence, trophallaxis appears to be more likely to be responsible for transfer than Ca. E. dacicola matings. Further research, such as the analysis of the proctodaeal diverticula and/or the crop system of lab flies after cohabitation with wild adults, together with behavioural studies, would better clarify this aspect. Moreover, cohabitation was the only treatment in which the endosymbiont was not that much exposed to oxygen. In contrast, the other treatment conditions, such as the olives, gelled water, eggs laid by wild females and faeces likely exposed to Ca. E. dacicola, were all exposed to oxygen for a longer period. We can therefore presume that Ca. E. dacicola prefers microaerophilic conditions for its vitality and transfer. In addition, we can affirm that transfer via cohabitation is not related to the sex of the wild symbiotic fly, since it occurred both when the Ca. E. dacicola contamination sources were wild females or wild males.
Hence, a symbiotic wild fly (male or female) in cohabitation with a non-symbiotic lab fly (male or female) is all that is required for the successful horizontal transfer of Ca. E. dacicola. Thus, this could be the first step in obtaining a permanently symbiotic laboratory olive fruit fly colony, likely reared on different substrates than the cellulose-based one, which allow for the avoidance of genetic modifications possibly caused by symbiont absence [19, 20].
The aim of the present study was to provide a reliable and consistent tool for implementing the detection of the endosymbiont in a large number of B. oleae specimens and/or environmental samples. According to the obtained results, it seems that the primers EdF1 and EdEnRev are not sufficiently specific for Ca. E. dacicola, as previously described by Estes et al. . Indeed, samples that were positive to Ca. E. dacicola with these primers did not show the same results after DGGE analysis. Moreover, an in silico analysis conducted using the Probe Match function within the RDP-II database (http://rdp.cme.msu.edu) showed a higher number of exact matches to the 16S rRNA gene sequences from members of Enterobacteriaceae family (3% respect to the total Enterobacteriaceae sequences in RDP database) belonging to Erwinia, Serratia, Proteus, Buttiauxella, Enterobacter and other genera. Thus, we suggest that to confirm the presence of Ca. E. dacicola, the screening of oesophageal bulbs or other specimens by PCR with EdF1/EdEnRev primer has to be combined with subsequent analyses . Sequencing is a time consuming and expensive method, and this does not seem to be the most convenient system, especially when a large number of samples must be analysed. ARDRA has been previously and successfully performed to compare profiles from uncultivable bulk bacteria residing in the oesophageal bulb with those from cultivable bacteria occasionally arising on plates in an attempt at endosymbiont isolation  and, more recently, to distinguish the two different bacterial haplotypes (htA and htB) . Furthermore, Ben-Yosef et al.  used DGGE performed with 986F-1401R primers and succeeded in detecting Ca. E. dacicola in B. oleae adult oesophageal bulbs and larvae. In this study, both ARDRA and DGGE techniques were applied. ARDRA demonstrated that it was possible to identify a specific profile corresponding to Ca. E. dacicola that was clearly distinguishable from that of other Enterobacteriaceae, such as M. morganii. Moreover, DGGE appears to be the best molecular fingerprinting method, since different bacterial taxa may be associated with oesophageal bulbs, both as individual dominant bacterium and in the bacterial consortium. The PCR-DGGE fingerprint was widely used to compare the microbial community structure in a variety of environments [43,44,45,46]. Furthermore, it supports the identification of bands, because PCR products can be recovered and sequenced . As an alternative to sequencing, the identification of bacteria may be achieved by the comparison of the PCR amplicon DGGE migration behaviour with that of a reference strain, used as species marker . Thus, the choice of which target hypervariable regions of the 16S rRNA gene are to be amplified may strongly affect the quality of information obtained by DGGE . This study demonstrated that PCR-DGGE performed with the primer set 63F-GC/518R and targeting the V1-V3 hypervariable regions, provides the best procedure for the rapid and straightforward screening of the presence of Ca. E. dacicola in a high number of fly specimens. This also reflects the two different Ca. E. dacicola haplotypes (htA and htB).
Considering the ARDRA profiles and the migration behaviour of PCR products on DGGE and nucleotide-sequence identity by BLAST, approximately 50% of the oesophageal bulbs of lab flies after cohabitation highlighted the presence of Ca. E. dacicola as a prominent associated species, and in particular, 13 corresponded to Ca. E. dacicola haplotype A and 13 to Ca. E. dacicola haplotype B, confirming previous findings from fly samples collected in Tuscany . Conversely, all the oesophageal bulbs of the lab-reared flies of the other crosses in the horizontal transfer experiment did not demonstrate the acquisition of Ca. E. dacicola. Furthermore, the other associated bacteria were supposed to be related to different taxa within the Enterobacteriaceae family.
The fact that M. morganii was detected in lab flies shows that the lab strain has been exposed to many bacteria and that M. morganii could have competed with Ca. E. dacicola, thus preventing horizontal transfer. This does not mean that M. morganii could represent a pathogen for B. oleae, as shown in recent studies on Anastrepha spp. [49, 50]. Furthermore, this bacterium has already been found in the oesophageal bulb of lab-reared B. oleae’s flies  and does not seem to represent a threat for the olive fruit fly. Along with this, supplementary observations would be appropriate to better evaluate the effects of this bacterium on B. oleae fitness and other parameters such as adult mortality or egg production.