Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for Dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila melanogaster model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.

Whole RNA, extracted from flies with or without Wolbachia infection, with or without SINV infection, was sequenced on an Illumina NextSeq. Resultant reads were mapped to existing genome assembiles for each entity and, using the RSEM workflow, quantified. Differential gene expression and splicing was assessed with EdegR.

Supplemental Table S1. Sequencing library metadata, statistics, and accession numbers.

Supplemental Table S2. Transcriptomic response to Wolbachia. DEGs are those with corrected P values < 0.05.

Supplemental Table S3. Transcriptomic response to SINV. DEGs are those with corrected P values < 0.05.

Supplemental Table S4. Transcriptomic response to SINV*TIME. DEGs are those with corrected P values < 0.05.

Supplemental Table S5. All normalized counts for all genes across all samples.

Supplemental Table S6. Genes with significant differences in isoform usage according to explanatory variable coefficients.

Supplemental Table S7. Genes with significant differences in isoform usage due to Wolbachia*SINV and/or Wolbachia*SINV*TIME along with functional annotations and notes to references in previously published literature.

Supplemental Table S8. Enriched terms in the joint STRING network.