Fig.4 Botryosphaeria dothidea cultivated on MEA medium. Growth of the mycelium after 5 days (a), 10 days (b) and 1 month (c) Barbora Zelinková - zelinkobar@natur.cuni.cz 1 - P. schultei 2 - A. echii 3 - L. arundinis (B) 4 - L. arundinis (A) 5 - L. arundinis (C) 6 - L. artemisiiae 7 - B. reticulatus . Fig.5 * PYSZKO, Petr, Hana ŠIGUTOVÁ, Miroslav KOLAŘÍK, Martin KOSTOVČÍK, Jan ŠEVČÍÍ K, Martin ŠIGUT, Denisa VIŠŇOVSKÁ and Pavel DROZD, 2024. Mycobiomes of two distinct cla des of ambro sia g all mid ges (Dipte ra: Cecidomyiid ae) are species-speci fic in lar vae but simi la r in nutritive mycelia. Microbiology Spectrum [online]. Available at: doi:10.1128/spectrum.02830-23 a b c Ambrosia gall midges (AGMs) are specialized Dipteran insects known for inducing galls, mostly on specific kind of host plants. These galls serve as intricate habitats where AGMs cultivate fungal symbionts in an ambrosia layer, a probable source of their nutrition. Additionally, AGMs may engage in phytomycetophagy, a unique feeding behaviour involving the consumption both of plant biomass and mycelium. The most commonly identified fungal species from mycelial layer is Botryosphaeria dothidea (Ascomycota: Dothideomycetes), which is known as a worldwide endophytic and pathogenic species threatening agricultural crops - currently Vitis vinifera. However, despite its frequent occurrence, B. dothidea has not been the subject of many studies on the significance of its secondary metabolites and their effect on the host plant, the insect larvae, or their possible involvement in gall initiation. Fig.1 - Asphondylia sarothamni on Cytisus Fig.2 - Opened gall of Asphondylia echii with pupa on Echium vulgare Fig.3 - Different stages of B. dothidea Fig.4 - Mycobiomes - Pyszko et al. (2024)* Fig.5 - Stable isotope compositions (preliminary data) To gain a better understanding of the interactions between fungi, larvae, and host plants, our next steps include determining the diversity and taxonomy of gall symbionts using metabarcoding (ITS for fungi, 16S for bacteria) followed by multilocus approach (TUB2, TEF1a) for problematic taxa. We will also explore the secondary metabolites, particularly those produced by Botryosphaeria dothidea, and their role in gall formation, insect development, and coexistence with other symbiotic fungi. Research on gall-associated fungi has primarily relied on cultivation methods, often neglecting potential larval endosymbionts. We used ITS2 rRNA metabarcoding to analyse the mycobiomes within different gall compartments (gall surface, gall interior, and larva) across six species from two phylogenetically distinct tribes (Asphondyliini and Lasiopterini) - Fig.4. The mycobiome of the gall interior was primarily dominated by Botryosphaeria dothidea (except in Lasioptera arundinis), confirming the long-standing hypothesis of its significant evolutionary association with AGMs. Feeding strategy of young Cecidomyiidaes is unknown for each particular species. The stable isotope compositions of carbon-13 (δ^13C) and nitrogen-15 (δ^15N) in Cecidomyiidae larvae (Asphondylia echii, Lasioptera artemisiiae, and Lasioptera arundinis) compared with those of strict phytophagous (Peruphasma schultei) and strict mycophagous (Bolitophagus reticulatus) species (Fig.5) distingues if their diet is leaning closer to phytophagous or mycophagous. ¹ Department of Biology and Ecology, Faculty of Science, University of Ostrava, Ostrava, Czech Republic ² Department of Zoology, Faculty of Science, Palacký University, Olomouc, Czech Republic ³ Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Fig.1 Fig.3 Petr Pyszko¹, Hana Šigutová¹'², Miroslav Kolařík³, Martin Kostovčík³, Jan Ševčík¹, Martin Šigut¹'³, Denisa Višňovská¹'³, Pavel Drozd¹, Eva Stodůlková³, Lenka Machová³, Barbora Zelinková³ Sincere thanks to the Czech Science Fund (GA23-07026S) for funding this project and Strategie AV21 project “VP33 MycoLife – the world of fungi” of