Wolbachia -mediated resistance to Zika virus infection in Aedes aegypti is dominated by diverse transcriptional regulation and weak evolutionary pressures

Abstract

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the w Mel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of w Mel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia -infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia . Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia -mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.

Author summary: When Wolbachia bacteria infect Aedes aegypti mosquitoes, they dramatically reduce the mosquitoes' susceptibility to infection with a range of arthropod-borne viruses, including Zika virus (ZIKV). Although this pathogen-blocking effect has been widely recognized, its mechanisms remain unclear. Furthermore, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to Wolbachia -mediated blocking. Here, we use host transcriptomics and viral genome sequencing to examine the mechanisms of ZIKV pathogen blocking by Wolbachia and viral evolutionary dynamics in Ae. aegypti mosquitoes. We find complex transcriptome patterns that do not suggest a single clear mechanism for pathogen blocking. We also find no evidence that Wolbachia exerts detectable selective pressures on ZIKV in coinfected mosquitoes. Together our data suggest that it may be difficult for ZIKV to evolve Wolbachia resistance, perhaps due to the complexity of the pathogen blockade mechanism.

Fig 1.. Vector competence of COL.wMel and COL.tet orally exposed to ZIKV-BC.

Mosquitoes were allowed to feed on ZIKV-BC-infected mice and were examined at days 4, 7, and 14 post-feeding to determine infection, dissemination, and transmission efficiencies. Infection efficiency corresponds to the proportion of mosquitoes with virus-infected bodies among the tested ones. Dissemination efficiency corresponds to the proportion of mosquitoes with virus-infected legs, and transmission efficiency corresponds to the proportion of mosquitoes with infectious saliva. *significant reduction in infection rates (Fisher’s Exact test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001) (A). 4 days post-feeding (Biological replicate number 1, n =30; Biological replicate number 2, n=30; Biological replicate 3, n=30) (B). 7 days post feeding (n=30; Biological replicate number 2, n=30; Biological replicate 3, n=30) (C). 14 days post feeding (n=30; Biological replicate number 2, n=No Data; Biological replicate 3, n=30).

Fig 2.. Transcriptional changes in COL.wMel relative to COL.tet.

(A) Volcano plots depicting differentially expressed transcripts in midgut and carcass tissues at 4 and 7 dpf, highlighting Aedes aegypti predicted innate immune genes (highlighted in magenta). Significant changes are transcripts with |log₂FoldChange| >1 (vertical red and blue dashed lines) and -log10(padj) > 0.05 (horizontal yellow dashed line). (B) Heatmap of differentially expressed mosquito innate immune genes in COL.wMel and COL.tet mosquitos that were exposed to an uninfected bloodmeal. The list of predicted innate immune genes was chosen from transcripts differentially regulated in COL.wMel carcass tissue 4dpf. (C) Transcripts with constitutively increased abundance (red) and decreased abundance (blue) across both collection time points. Transcripts with a difference in log ₂ FoldChange > 2.5 and that are annotated in VectorBase are labeled. Muscle lim protein (VB ID: AAEL019799). GPRMTH6 (VB ID: AAEL011521). No genes went from being significantly increased to significantly decreased or vice versa between 4 dpf and 7dpf.

Fig 3.. Gene Ontology analysis of COL.wMel midguts 4 days post blood feeding.

GO terms associated with the differentially expressed transcripts in COL.wMel midguts at 4dpf. The top 10 GO terms from each category (Biological Process, Cellular Component, Molecular Function), determined by topGO, were run in the GO Figure! pipeline to combine semantically similar terms and reduce redundancy. Terms are ranked by lowest log10(p-value). The size of each graphical point corresponds to the number of topGO terms associated with the listed summarizing term.

Fig 4:. Transcriptional changes in COL.wMel relative to COL.tet following a ZIKV-infected bloodmeal.

(A) Volcano plots showing differentially expressed transcripts in midgut and carcass tissues at 4 and 7 dpf after a ZIKV-infected bloodmeal, highlighting Aedes aegypti predicted innate immune genes (highlighted in magenta). Significant changes are transcripts with |log2FoldChange| >1 (vertical red and blue dashed lines) and -log10(padj) > 0.05 (horizontal yellow dashed line). Aedes aegpyti predicted innate immune genes are highlighted in magenta. (B) Heatmap of mosquito innate immune gene expression patterns in ZIKV-exposed COL.wMel and COL.tet mosquitos. The list of predicted innate immune genes was chosen from transcripts differentially regulated in COL.wMel carcass tissue 4dpf. (C) Transcripts with constitutively increased abundance (red) and decreased abundance (blue) between both collection time points. Transcripts with a difference in log2FoldChange > 2.5 are labeled and that are characterized in VectorBase are labeled. Alpha-actinin (AAEL007306). No genes went from being significantly increased to significantly decreased or vice versa between 4 dpf and 7dpf.

Fig 5.. Gene ontology analysis in COL.wMel midguts 4 days post feeding on ZIKV-infected mice.

GO terms associated with the differentially expressed transcripts in COL.wMel relative to COL.tet midguts at 4dpf on ZIKV-infected mice. The top 10 GO terms from each category (Biological Process, Cellular Component, Molecular Function), determined by topGO, were run in the GO Figure! pipeline to combine semantically similar terms and reduce redundancy. Terms are ranked by lowest log10(p-value). The size of each graphical point corresponds to the number of topGO terms associated with the listed summarizing term.

Fig. 6.. The induced response to ZIKV is distinct in COL.wMel compared to COL.tet.

The correlation in log ₂ fold change between COL.wMel and COL.tet is shown at 4 and 7 days post feeding in carcass and midgut tissues. The transcripts highlighted in color differ in the direction or magnitude of expression (interaction term significant at p<0.01). Blue indicates transcripts unique to COL.wMel, orange indicates transcripts unique to COL.tet, and green indicates transcripts that are shared between the two mosquito types.

Fig 7.. Differentially expressed transcripts and gene ontology analysis in COL.tet midguts 7 days post-feeding on ZIKV-infected mice.

(A). Volcano plots demonstrating differentially expressed transcripts in the midgut of COL.tet midguts. Significant changes are transcripts with |log2FoldChange| >1 (vertical red and blue dashed lines) and -log10(p-adjusted) > 0.05 (horizontal yellow dashed line). (B). GO terms associated with transcripts with an increased abundance in COL.tet midguts 7dpf. Terms are ranked by lowest log10(p-value). The size of each graphical point corresponds to the number of topGO terms associated with the listed summarizing term.

Fig 8.. Zika virus is under weak purifying selection in Wolbachia-infected and Wolbachia-free mosquitoes.

(A). Per-sample nucleotide diversity is quantified for nonsynonymous (πN; orange) and synonymous (πS; blue) sites across all ZIKV plaque-positive tissues collected from COL.wMel and COL.tet mosquitoes. (B). The difference between πN and πS is plotted against the null hypothesis of neutral selection, denoted by a horizontal black line at zero. (C). Barcode species richness was quantified as the number of unique barcode species detected in each sample. All groups underwent 10,000 Bayesian bootstrap replicates, from which mean values and standard deviations were calculated.