RNA-barcoding: rapid and reliable characterization of fungal symbiont communities in insects

The ambrosia symbiosis between wood boring beetles and fungi is an emerging model system in symbiology. Invasive ambrosia beetles and fungi are also becoming a major threat to the world’s forests and tree industries. Current methods for fungus community characterization are not sufficient to rapidly and reliably characterize this hyper-diverse as well as important symbiosis. Culturing is slow and requires live samples, high-throughput metabarcoding suffers from biases.

Here we report a new method for symbiont community characterization based on rRNA reverse-transcription and sequencing. The method reduces bias by only recovering actively metabolizing fungi in the mycangium while excluding dormant contaminants present as spores. Mycangia are extracted from beetles, flash-frozen and subjected to RNA extraction. Reverse transcriptase is used to create a cDNA library for a 402bp section of LSU rRNA bounded by a universal ribosomal DNA primer pair. Libraries are PCR-amplified by fusion primers and sequenced on Illumina MiSeq. Our data show sensitivity to 10 fg/µl of RNA (a few fungal cells), and significantly greater recovery of living relevant symbionts compared to traditional metabarcoding.