Untangling an insect's virome from its endogenous viral elements

Abstract

Background: Insects are an important reservoir of viral biodiversity, but the vast majority of viruses associated with insects have not been discovered. Recent studies have employed high-throughput RNA sequencing, which has led to rapid advances in our understanding of insect viral diversity. However, insect genomes frequently contain transcribed endogenous viral elements (EVEs) with significant homology to exogenous viruses, complicating the use of RNAseq for viral discovery.

Methods: In this study, we used a multi-pronged sequencing approach to study the virome of an important agricultural pest and prolific vector of plant pathogens, the potato aphid Macrosiphum euphorbiae. We first used rRNA-depleted RNAseq to characterize the microbes found in individual insects. We then used PCR screening to measure the frequency of two heritable viruses in a local aphid population. Lastly, we generated a quality draft genome assembly for M. euphorbiae using Illumina-corrected Nanopore sequencing to identify transcriptionally active EVEs in the host genome.

Results: We found reads from two insect-specific viruses (a Flavivirus and an Ambidensovirus) in our RNAseq data, as well as a parasitoid virus (Bracovirus), a plant pathogenic virus (Tombusvirus), and two phages (Acinetobacter and APSE). However, our genome assembly showed that part of the 'virome' of this insect can be attributed to EVEs in the host genome.

Conclusion: Our work shows that EVEs have led to the misidentification of aphid viruses from RNAseq data, and we argue that this is a widespread challenge for the study of viral diversity in insects.

Keywords: Aphids; Endogenous viral elements; Insects; RNAseq; Viral discovery.

Fig. 1

Details of the per sample breakdown of reads aligning to specific bacterial (A), eukaryotic (B), and viral (C) taxa. Reads per million aligned to the nucleotide database (NT rPM) was used as the quantitative metric in the heatmaps (see Table S5 for metric details)

Fig. 2

Assembled M. euphorbiae transcriptome contigs aligning to previously a described insect Flavivirus (A) and an Ambidensovirus (B)

Fig. 3

Frequency of Macrosiphum euphorbiae virus 1 (MeV-1), Macrosiphum euphorbiae virus 2 (MeV-2), and Hamiltonella denfesa infections in wild-collected (n = 23) and Me57 laboratory established (n = 1) aphids. All samples were tested using cDNA from individual aphids for PCR screenings

Fig. 4

M. euphorbiae genome assembly metrics (A), GC content and coverage (B), and BUSCO metrics (C)