LB 4-32 - Diptera diversity in Tuber aestivum ascomata

Fungi of the genus Tuber are ectomycorrhizal ascomycetes that live in association with the roots of many plant species. As Tuber spp. produce hypogeous ascomata, they are strictly dependent on animals for the dispersal of their spores. The list of animals that feed on truffles is far from complete and the role of each species in the life cycle of truffles is largely unknown. In the case of arthropods, mainly in the orders Coleoptera and Diptera, most of the studies have analyzed only single species that feed and reproduce on a few truffle ascomata. We assume that the diversity of arthropods feeding on truffles is largely underestimated. In this study, we molecularly characterized larvae and adults of Diptera developing in 23 ascomata of T. aestivum with obvious symptoms of infestation. They were collected from natural and cultivated truffle sites in two Italian provinces: L’Aquila (central Italy) and Bologna (northern Italy). Twenty-three larvae were selected from 16 ascomata within 24 hours of harvest and stored in 95% ethanol until DNA extraction. The other seven ascomata were placed in glass jars containing sterile sand and kept in the dark at room temperature until adult emergence. The adults recovered from each ascoma (109 in total) were also stored in 95% ethanol and DNA was extracted from 28 of these. The standard barcode region of the mitochondrial cytochrome c oxidase I gene was amplified. The sequences were first compared with those in the GenBank and Bold databases and then clustered using the tclust function of the R package spider v1.5.0 with different thresholds (2%, 3.5%,5%, 8%). The optimal threshold was 3.5% (obtained using the localMinima function), which divided the sequences into 7 clusters. About 50% of the samples (larvae or adults) belonging to 4 different lineages were identified at the family level (Helomyzidae), while Cheilosia soror (22 samples, 7 truffles), Drosophila subobscura (2 samples, 1 truffle) and Phaonia trimaculata (2 samples, 2 truffles) were identified as species. Cheilosia soror and D. subobscura were found in truffles from both sampling areas. Several species of the genus Suillia and the family Phoridae have been described in association with T. aestivum, but none of the species identified in this work have been reported previously. The prospects of this type of research aim to extend the sampling as well as a wider geographical area also for different species of Tuber.