# Niche Differentiation and Predicted Functions of Microbiomes in a Tri-Trophic Willow–Gall (Euura viminalis)–Parasitoid Wasp System

## TL;DR

This study explores how the microbiomes of a willow-gall-parasitoid system differ based on their niches and what functions they perform.

## Contribution

The paper reveals niche-specific patterns and predicted functions of bacterial and fungal communities in a tri-trophic system.

## Key findings

- Fungal communities were richer in open, air-exposed sites, while bacteria thrived in sealed gall interiors.
- Fungi showed positive connectivity in exposed niches, while bacteria had negative associations in enclosed environments.
- Microbiome patterns suggest roles in transport, metabolism, and biocontrol in different habitats.

## Abstract

Symbiotic microbes support insect survival and development, yet how niches shape them in a willow–gall–parasitoid system is unclear. We sampled six types of sites—leaf and gall surfaces, gall interiors, sawfly larvae, and wasps—and used genetic sequencing to identify bacteria and fungi. A clear pattern emerged: open, air-exposed sites (leaf and gall surfaces and wasps) held richer, more balanced fungal communities, whereas the sealed gall interiors and larvae contained few fungi. Bacteria showed the opposite pattern, with the highest diversity inside galls. Habitats organized these communities more than host identity, informing more precise, microbe-aware pest control programs.

Chalcidoids (Hymenoptera: Chalcidoidea), the most important natural enemies of parasitoids, serve as a pivotal factor in the regulation and management of pest populations. Microbiotas mediate interactions among plants, herbivores, and natural enemies and shape host immunity, parasitoid development, and gall formation; however, the niche-specific diversity and functions of tritrophic parasitoid–host–gall systems remain unclear. Focusing on leaf galls induced on twisted willow (Salix matsudana f. tortuosa) by the willow-galling sawfly Euura viminalis and on two chalcidoids, Eurytoma aethiops and Aprostocetus sp., we profiled bacterial and fungal microbiomes across plant surfaces, gall lumen, host larval tissues, and parasitoids using HTAS. Fungal diversity peaked on parasitoids but was depleted in the gall lumen and host tissues; bacterial richness showed the opposite trend, peaking in the gall lumen and decreasing on parasitoids. In networks contrasted by kingdom, fungi showed positive interface-hub connectivity (Cladosporium, Alternaria), whereas bacteria showed negative hub-mediated associations (Pseudomonas, Acinetobacter), indicating habitat-specific replacements: exposed niches favored transport, two-component, secretion–motility and energy functions, whereas the gall lumen reduced transport/motility but selectively retained N/S metabolism; and in host tissues, information processing and nitrogen respiration were highlighted. These results inform microbiome-guided parasitoid biocontrol.