Niche Differentiation and Predicted Functions of Microbiomes in a Tri-Trophic Willow-Galls (Euura viminalis)-Parasitoid wasps System

Chalcidoid wasps (Hymenoptera: Chalcidoidea), the most important parasitoid natural enemies, serving as a pivotal factor in the regulation of pest populations and advancing sustainable pest management. Microbiota mediate interactions among plants, herbivores, and natural enemies, shaping host immunity, parasitoid development, and gall formation, yet niche-specific diversity and functions in tritrophic parasitoid-host-gall systems remain unclear. Focusing on leaf galls induced by the willow-galling sawfly Euura viminalis (Linnaeus, 1758) on twisted willow (Salix matsudana f. tortuosa), and two chalcidoids-Eurytoma aethiops (Eurytomidae) and Aprostocetus sp. (Eulophidae)-we profiled bacterial (16S) and fungal (ITS) microbiomes across plant surfaces, gall lumen, host larval tissues, and parasitoids using high-throughput amplicon sequencing. Fungal diversity peaked on parasitoids but was depleted in gall lumen and host tissues; bacterial richness showed the opposite trend, peaking in the gall lumen and lower on parasitoids. Networks contrasted by kingdom: fungi showed positive, interface-hub connectivity (Cladosporium, Alternaria, Acremonium), whereas bacteria showed hub-mediated negative associations (Pseudomonas, Acinetobacter, Pantoea) marking habitat-specific replacements. Predicted functions tracked a habitat-openness axis: exposed niches enriched for transport/two-component/secretion-motility and energy pathways; the gall lumen showed reduced transport/motility with selective N/S features; host tissues highlighted information processing and nitrogen respiration. These results motivate shotgun/transcriptomic and enzymatic validation and inform microbiome-guided parasitoid biocontrol.