Inside the Belly of the Beast: Exploring the Gut Bacterial Diversity of Gonipterus sp. n. 2

Abstract

The Eucalyptus snout beetle (Gonipterus sp. n. 2) is a destructive invasive pest of Eucalyptus plantations, responsible for significant defoliation and wood yield losses globally. Native to Australia, this beetle has adapted to thrive on diverse Eucalyptus hosts, overcoming their chemical defences. However, the mechanisms by which Gonipterus tolerates or utilises these plant defence metabolites remain poorly understood. In South Africa, Gonipterus sp. n. 2 poses a significant threat to Eucalyptus plantations by causing extensive defoliation and leading to substantial reductions in growth and wood production. This study investigates the relationship between diet, host Eucalyptus species, and the gut microbiome of Gonipterus sp. n. 2. Using controlled feeding experiments, beetles were reared on artificial, semi-artificial, and natural diets, as well as two Eucalyptus genotypes with distinct secondary metabolite profiles. High-throughput 16S rDNA sequencing and gas chromatography-mass spectrometry (GC-MS) revealed significant shifts in gut bacterial diversity and composition across diets. Natural diets supported the most diverse microbial communities, while artificial diets fostered a homogenised microbiome dominated by opportunistic taxa like Serratia. Host-specific effects were observed in frass microbiota, with substantial biotransformation of monoterpenes into less toxic derivatives. The results highlight the plasticity of Gonipterus gut microbiota, which enables metabolic adaptability and resilience in diverse environments. This microbial flexibility underpins the invasiveness of Gonipterus, emphasising the role of gut symbionts in overcoming host chemical defences. Understanding these interactions offers novel insights for microbiome-targeted pest management strategies, providing a sustainable approach to mitigate the impact of Gonipterus on global Eucalyptus forestry.

Keywords: Eucalyptus snout beetle; Insect-microbe interactions; Invasive pests; Microbiome; Plant metabolites.

Fig. 1

Experimental workflow to study the impact of various dietary treatments on bacterial biodiversity and plant metabolite breakdown within the gut of the Eucalyptus pest Gonipterus sp. n. 2

Fig. 2

Effects of diet and rearing environment on gut-associated bacterial biodiversity in Gonipterus sp. n. 2. A Taxonomic composition of bacteria in the gut up to order level. Numerical within the parenthesis beside taxon names indicate read number; B Venn diagram showing the number of shared and unique OTUs between the three diet and rearing environment sub-groups; D Shannon index, E Simpson index, F OTU richness (Chao1), and (G) principal coordinates analysis. Analyses of α and β diversity were conducted in Microbiomeanalyst 2.0. α diversity (Shannon, Simpson) and species richness (Chao1) were assessed on original data using Kruskal–Wallis tests. β diversity was analysed with normalised data using a Bray–Curtis based PCoA and evaluated with PERMANOVA and PERMDISP

Fig. 3

Foliar volatile content of Eucalyptus genotypes and Gonipterus frass. (A) Relative GCMS intensities of metabolites between plant and frass groups (B) Relative GC–MS peak intensities of volatile organic compounds in leaves of two Eucalyptus genotypes: Eucalyptus dunnii and Eucalyptus grandis × Eucalyptus urophylla. (C) Relative GC–MS peak intensities of volatile organic compounds in beetle frass after beetles consumed E. dunnii or E. grandis × E. urophylla host leaves. (D, E) Heatmap showing the relative concentrations of specific monoterpenes in Eucalyptus leaves and their biotransformation products in beetle frass after beetles were fed on leaves from E. dunnii and E. grandis × E. urophylla hosts. Data were normalised and log-transformed in Metaboanalyst 6.0 for PCoA generation. Additional processing and deconvolution of Eucalyptus genotypes and the associated frass samples were conducted in Mass Hunter, followed by normalisation and heatmap generation to compare the relative concentrations of metabolites across the samples

Fig. 4

Radial plot illustrating the taxonomic composition of bacteria in the gut and frass of Gonipterus sp. n. 2 after feeding on E. dunnii and E. grandis × E. urophylla. The plot was generated in Flourish. The numerical within the parenthesis beside each taxon indicates the read number. Overlapping taxa are marked with purple bullets

Fig. 5

Effects of feeding on E. dunnii and E. grandis × E. urophylla leaves on gut and frass-associated bacterial biodiversity in Gonipterus sp. n. 2. gut (A–C) α diversity indices; D principal coordinates analysis. Frass (E–G) α diversity indices; H principal coordinates analysis. Venn diagrams illustrating shared and unique OTUs between (I) the gut of beetles fed with Eucalyptus dunnii or E. grandis × E. urophylla, (J) the frass of beetles fed with E. dunnii or E. grandis × E. urophylla, K the gut and frass of beetles fed with E. dunnii. L The gut and frass of beetles fed with E. grandis × E. urophylla. M The gut of beetles fed with E. dunnii, E. grandis × E. urophylla, or the ‘natural’ sub-group from the field (see Fig. 1). α and β diversity analyses were performed using MicrobiomeAnalyst 2.0. α diversity was assessed using original data and Mann–Whitney tests. β diversity was analysed using a Bray–Curtis-based PCoA on filtered and normalised data, with statistical significance evaluated with PERMANOVA and PERMDISP