Tuber spp. (Ascomycota) forms hypogeous fruiting bodies (truffles) that host many microbial species as well as invertebrates which feed on them. Despite the larvae and adults of Diptera and Coleoptera are commonly found to inhabit truffles, molecular investigations assessing their occurrence are still few and the number of species is probably underestimated. In this study, 52 larvae and adults of Diptera from 23 T. aestivum ascomata collected in two provinces of northern and central Italy were molecularly characterized. The sequences fell into four Diptera families, and four taxa were identified as Cheilosia soror, Phaonia cf. trimaculata, Drosophila subobscura, and Suillia gigantea. Morphology of adults belonging to these species confirmed their identity. Additional three taxa belonging to the Helomyzidae remained unclassified. The study highlighted the coexistence of different Diptera species in the same ascoma, suggesting potential lack of competitive exclusion. Geographical distribution analysis reveals non-site specificity for most species. This research contributes insights into the diversity of Dipteran species and their interactions with truffles and lays the groundwork for their monitoring, at a time where truffle resources are threatened by anthropic and environmental factors.
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