Wood fibers are a crucial microhabitat for cellulose- and xylan- degrading bacteria in the hindgut of the wood-feeding beetle Odontotaenius disjunctus

Abstract

Introduction: Wood digestion in insects relies on the maintenance of a mosaic of numerous microhabitats, each colonized by distinct microbiomes. Understanding the division of digestive labor between these microhabitats- is central to understanding the physiology and evolution of symbiotic wood digestion. A microhabitat that has emerged to be of direct relevance to the process of lignocellulose digestion is the surface of ingested plant material. Wood particles in the guts of some termites are colonized by a specialized bacterial fiber-digesting microbiome, but whether this represents a widespread strategy among insect lineages that have independently evolved wood-feeding remains an open question.

Methods: In this study, we investigated the bacterial communities specifically associated with wood fibers in the gut of the passalid beetle Odontotaenius disjunctus. We developed a Percoll-based centrifugation method to isolate and enrich the wood particles from the anterior hindgut, allowing us to access the wood fibers and their associated microbiome. We then performed assays of enzyme activity and used short-read and long-read amplicon sequencing of the 16S rRNA gene to identify the composition of the fiber-associated microbiome.

Results: Our assays demonstrated that the anterior hindgut, which houses a majority of the bacterial load, is an important site for lignocellulose digestion. Wood particles enriched from the anterior hindgut contribute to a large proportion of the total enzyme activity. The sequencing revealed that O. disjunctus, like termites, harbors a distinct fiber-associated microbiome, but notably, its community is enriched in insect-specific groups of Lactococcus and Turicibacter.

Discussion: Our study underscores the importance of microhabitats in fostering the complex symbiotic relationships between wood-feeding insects and their microbiomes. The discovery of distinct fiber-digesting symbionts in O. disjunctus, compared to termites, highlights the diverse evolutionary paths insects have taken to adapt to a challenging diet.

Keywords: Passalidae; beetles; gut microbiomes; lignocellulose; symbiotic digestion.

FIGURE 1

Schematic diagram (A) of the gut compartments of Odontotaenius disjunctus analyzed for bacterial density (B) and enzyme (cellulase and xylanase) activity (C). Bacterial density in the compartments is represented as normalized counts of qPCR-amplified 16S rRNA genes. One unit of enzyme activity is defined as 1 μmol of sugar equivalent released per minute, per gram of insect. **marks significant differences (P ≤ 0.001).

FIGURE 2

Scanning electron micrograph of bacterial cells adhering to wood fibers in the anterior hindgut of O. disjunctus (A) and rough degradation of the wood fiber surface (B). Arrows point to areas with likely fungal degradation.

FIGURE 3

Histogram of wood fiber lengths in the AHG lumen of O. disjunctus and in the fiber fraction obtained from Percoll density-gradient centrifugation of luminal fluid.

FIGURE 4

Schematic representation (A) of the Percoll fractionation of the luminal content in the AHG of O. disjunctus into the Fiber-free and Fiber fractions, cellulase activity (B), xylanase activity (C), and bacterial density (D). Bacterial density in the compartments is represented as normalized counts of qPCR-amplified 16S rRNA genes. One unit of enzyme activity is defined as 1 μmol of sugar equivalent released per minute, per gram of insect. **and mark significant differences at the P ≤ 0.001 and P ≤ 0.05 levels.

FIGURE 5

Non -metric Multidimensional Scaling (NMDS) analysis of weighted UniFrac distances between bacterial communities associated with AHG luminal content, the fiber and fiber-free fractions obtained from Percoll density gradient centrifugation.

FIGURE 6

Relative abundance of bacterial genera differentially abundant between the fiber and fiber-free community across all samples. *Termite Treponema cluster.

FIGURE 7

Consensus trees based on partial 16S rRNA genes for 1,000 ultrafastbootstrap replicates of Lactococcus (calculated with 129 sequences and 1,570 columns) and Turicibacter (calculated with 681 sequences and 1,590 columns). Nodes showing less than 60% support have been collapsed into multifurcations. Edges and circles are, respectively colored by diet and taxonomy of the hosts, from which the sequences were obtained.