Ambrosia gall midges are endophagous insect herbivores whose larvae live enclosed within a single gall for their entire development period. They may exhibit phytomycetophagy, a remarkable feeding mode that involves the consumption of plant biomass and mycelia of their cultivated gall symbionts. Thus, AGMs are ideal model organisms for studying the role of microorganisms in the evolution of host specificity in insects. However, compared to other fungus-farming insects, insect-fungus mutualism in AGMs has been neglected. Our study is the first to use DNA metabarcoding to characterize the complete mycobiome of the entire system of the gall-forming insects as we profiled gall surfaces, nutritive mycelia, and larvae. Interestingly, larval mycobiomes were significantly different from their nutritive mycelia, although Botryosphaeria dothidea dominated the nutritive mycelia, regardless of the evolutionary separation of the tribes studied. Therefore, we confirmed a long-time hypothesized paradigm for the important evolutionary association of this fungus with AGMs.

• Keywords
• Asphondylia, Cecidomyiidae, Lasioptera, ambrosia gall midge, fungiculture, larval mycobiome, metabarcoding, nutritive mycelium, phytomycetophagy,
• MeSH
• Ambrosia MeSH
• Diptera * MeSH
• Insecta MeSH
• Larva MeSH
• Mycobiome * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

The caterpillar gut is an excellent model system for studying host-microbiome interactions, as it represents an extreme environment for microbial life that usually has low diversity and considerable variability in community composition. Our study design combines feeding caterpillars on a natural and artificial diet with controlled levels of plant secondary metabolites and uses metabarcoding and quantitative PCR to simultaneously profile bacterial and fungal assemblages, which has never been performed. Moreover, we focus on multiple caterpillar species and consider diet breadth. Contrary to many previous studies, our study suggested the functional importance of certain microbial taxa, especially bacteria, and confirmed the previously proposed lower importance of fungi for caterpillar holobiont. Our study revealed the lack of differences between monophagous and polyphagous species in the responses of microbial assemblages to plant secondary metabolites, suggesting the limited role of the microbiome in the plasticity of the herbivore diet.

• Keywords
• bacterial and fungal microbiomes, invertebrate–microbe interactions, network stability, plant secondary metabolite, salicylic acid, tannin, tannivin,
• MeSH
• Bacteria genetics   MeSH
• Microbiota * MeSH
• Mycobiome * MeSH
• Plants MeSH
• Publication type
• Journal Article MeSH

Microorganisms are key mediators of interactions between insect herbivores and their host plants. Despite a substantial interest in studying various aspects of these interactions, temporal variations in microbiomes of woody plants and their consumers remain understudied. In this study, we investigated shifts in the microbiomes of leaf-mining larvae (Insecta: Lepidoptera) and their host trees over one growing season in a deciduous temperate forest. We used 16S and ITS2 rRNA gene metabarcoding to profile the bacterial and fungal microbiomes of leaves and larvae. We found pronounced shifts in the leaf and larval microbiota composition and richness as the season progressed, and bacteria and fungi showed consistent patterns. The quantitative similarity between leaf and larval microbiota was very low for bacteria (~9%) and decreased throughout the season, whereas fungal similarity increased and was relatively high (~27%). In both leaves and larvae, seasonality, along with host taxonomy, was the most important factor shaping microbial communities. We identified frequently occurring microbial taxa with significant seasonal trends, including those more prevalent in larvae (Streptococcus, Candida sake, Debaryomyces prosopidis, and Neoascochyta europaea), more prevalent in leaves (Erwinia, Seimatosporium quercinum, Curvibasidium cygneicollum, Curtobacterium, Ceramothyrium carniolicum, and Mycosphaerelloides madeirae), and frequent in both leaves and larvae (bacterial strain P3OB-42, Methylobacterium/Methylorubrum, Bacillus, Acinetobacter, Cutibacterium, and Botrytis cinerea). Our results highlight the importance of considering seasonality when studying the interactions between plants, herbivorous insects, and their respective microbiomes, and illustrate a range of microbial taxa persistent in larvae, regardless of their occurrence in the diet. IMPORTANCE Leaf miners are endophagous insect herbivores that feed on plant tissues and develop and live enclosed between the epidermis layers of a single leaf for their entire life cycle. Such close association is a precondition for the evolution of more intimate host-microbe relationships than those found in free-feeding herbivores. Simultaneous comparison of bacterial and fungal microbiomes of leaves and their tightly linked consumers over time represents an interesting study system that could fundamentally contribute to the ongoing debate on the microbial residence of insect gut. Furthermore, leaf miners are ideal model organisms for interpreting the ecological and evolutionary roles of microbiota in host plant specialization. In this study, the larvae harbored specific microbial communities consisting of core microbiome members. Observed patterns suggest that microbes, especially bacteria, may play more important roles in the caterpillar holobiont than generally presumed.

• Keywords
• bacteria, fungi, invertebrate-microbe interactions, microbial communities, microbial ecology, plant-microbe interactions,
• MeSH
• Bacteria genetics   MeSH
• Diet MeSH
• Larva MeSH
• Mycobiome * MeSH
• Seasons MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH