# Coevolution and Functional Effects of Endosymbiotic Rickettsia in Leptocybe invasa Fisher & LaSalle (Hymenoptera: Eulophidae) Across China

## TL;DR

This study explores how Rickettsia bacteria coevolve with the invasive wasp Leptocybe invasa in China and how they affect the wasp's biology and reproduction.

## Contribution

The discovery of two distinct Rickettsia strains and their specific associations with host lineages, along with transcriptomic insights into symbiont effects.

## Key findings

- Rickettsia infection is 100% prevalent in L. invasa populations across China.
- Tetracycline treatment eliminates Rickettsia and alters L. invasa's sex ratio and lifespan.
- Transcriptome analysis reveals 178 differentially expressed genes linked to Rickettsia presence.

## Abstract

Rickettsia is an endosymbiotic bacterium that infects various arthropods, affecting the host's biology, ecology, and evolution. Leptocybe invasa is an invasive pest that severely damages eucalyptus plants. A comprehensive investigation of Rickettsia in 313 female L. invasa individuals from 17 Chinese populations revealed a 100% infection prevalence. Sequencing of three host molecular markers—mitochondrial COI, nuclear ITS, and 28S—led to the identification of a novel L. invasa haplotype, designated Haplotype 1 × 2, which exhibits mito‐nuclear discordance. Concurrently, sequencing of four Rickettsia genes (16S rRNA, gltA, atpA, rpmE) revealed two distinct strains, termed STRiA and STRiB. These strains demonstrated a specific association with the host lineages, where STRiA was exclusively associated with lineage A (comprising Haplotype 1 and Haplotype 1 × 2), and STRiB was linked to lineage B. Phylogenetic analysis of the multigene datasets from both the host and Rickettsia revealed a high degree of topological congruence between their inferred trees. Correlation analysis further demonstrated a moderate positive association (r = 0.307). The significance of this relationship was supported by a Mantel test (p < 0.005), suggesting coevolution. Low‐dose tetracycline treatment effectively eliminated Rickettsia from L. invasa. L. invasa treated with tetracycline exhibited a significantly higher proportion of male offspring, reduced Rickettsia expression, and decreased body length and lifespan in female offspring. Transcriptome analysis comparing Rickettsia‐free and Rickettsia‐infected L. invasa following antibiotic treatment identified 178 differentially expressed genes (122 up‐regulated, 56 down‐regulated). These genes were enriched in GO terms related to metabolic processes, cellular processes, cellular components, binding functions, and catalytic activities. KEGG pathway analysis revealed enrichment of differentially expressed genes primarily in metabolic pathways, insect hormone biosynthesis, and thermogenesis. Additionally, enrichment was observed in key signaling pathways, including Ras, MAPK, NF‐κB, TGF‐β, TNF, and Apelin. These findings elucidate the coevolutionary relationship and functional roles of Rickettsia in L. invasa, providing a foundation for symbiont‐mediated biological control.

Two distinct lineages of L. invasa and the dominant endosymbiont, Rickettsia, was identified, followed by an analysis of its infection density across various developmental stages and tissues of L. invasa. Furthermore, two distinct strains of the Rickettsia endosymbiont were detected, and the phylogenetic relationships between these strains and their L. invasa hosts were characterized. L. invasa was treated with tetracycline. The effects of Rickettsia infection on the reproduction, offspring sex ratio, and lifespan of L. invasa were investigated. Finally, transcriptome sequencing was performed to reveal genetic changes in L. invasa following antibiotic‐mediated removal of Rickettsia and to explore the functional mechanisms of this endosym‐biont within its host.