Differing Transcriptomic Responses in High Titer versus Low Titer Aedes aegypti Mosquitoes after Oral Infection with Sindbis Virus

Abstract

Oral infection of mosquitoes by arboviruses often results in a large degree of variation in the amount of infectious virus between individual mosquitoes, even when the mosquitoes are from inbred laboratory strains. This variability in arbovirus load has been shown to affect virus transmissibility. Previously, our group described population genetic and specific infectivity differences between the virus populations found in high and low titer Aedes aegypti mosquitoes that had been orally infected with Sindbis virus (SINV). In this study, we sought to investigate whether there were also differences in transcriptomic response between these high and low titer mosquitoes. Results from the transcriptomic data analysis showed that more genes involved in antiviral activity, endopeptidase activity, and methyltransferase activity were upregulated in low titer mosquitoes than in high titer mosquitoes, relative to blood-fed controls. Meanwhile, genes involved in ion transport, energy metabolism, acetylation, glycosylation, lipid metabolism, and transport tended to be upregulated in high titer mosquitoes more than in low titer mosquitoes, relative to blood-fed mosquitoes. Overall, genes involved in antiviral activities tended to be upregulated in low titer mosquitoes while genes involved in proviral activities were mostly upregulated in high titer mosquitoes. This study has identified a number of candidate mosquito genes that are putatively associated with SINV titer variability after oral infection of Ae. aegypti, and these can now be investigated in order to ascertain their roles in virus replication and their contributions to determining vector competence.

Keywords: Aedes aegypti; Sindbis virus; arbovirus; mosquito; vector competence.

Figure 1

A schematic diagram showing the experimental setup used in this study. SINV was mixed with sheep blood and used to orally infect 3-day-old mosquitoes. At 5 days post blood meal, the mosquitoes were dissected into midguts and carcasses. Portions of the carcass samples were used to determine the titer of SINV in order to identify low titer and high titer mosquitoes. Poly(A)⁺ RNA was then isolated from the midguts and the remaining portions of the carcass samples from six low titer and six high titer samples, as well as from the midguts and carcasses of three control mosquitoes that were fed only blood, and the RNA was used to generate sequencing libraries that were then subjected to Illumina sequencing and analysis. The figure was generated using BioRender (https://www.biorender.com) (accessed on 17 September 2024). Some of the images in the figure were previously published [22].

Figure 2

(A) Principal component analysis and (B) hierarchy cluster analysis of the transcriptomic data from six high titer mosquitoes, six low titer mosquitoes, and three blood fed mosquitoes. BFC, blood-fed carcass; BFM, blood-fed midgut; HTC, high titer carcass; HTM, high titer midgut; LTC, low titer carcass; LTM, low titer midgut. Each dot in (A) represents a single mosquito.

Figure 3

Volcano plot analysis of DEGs observed in (A) low titer midguts, (B) high titer midguts, (C) low titer carcasses, and (D) high titer carcasses (all relative to blood-fed ones). RefSeq identifiers are provided for the top five upregulated or downregulated DEGs in each category.

Figure 3

Volcano plot analysis of DEGs observed in (A) low titer midguts, (B) high titer midguts, (C) low titer carcasses, and (D) high titer carcasses (all relative to blood-fed ones). RefSeq identifiers are provided for the top five upregulated or downregulated DEGs in each category.

Figure 4

Venn diagram showing the numbers of DEGs with ≥two-fold expression difference (compared to blood-fed ones) that were unique or shared between low titer carcasses (LTC), low titer midguts (LTM), high titer carcasses (HTC), and high titer midguts (HTM).

Figure 5

Heatmaps of DEGs involved in (A) antiviral activity, (B) ion transport, (C) lipid metabolism and transport, (D) energy metabolism, (E) peptidase activity, and (F) epigenetic modification. The averages of the gene matrix data were log₂ transformed before they were used to generate the heatmaps.

Figure 6

GO term analysis of (A) molecular functions of upregulated DEGs in midguts of low titer and high titer mosquitoes, (B) molecular functions of upregulated DEGs in carcasses of low titer and high titer mosquitoes, (C) biological processes of upregulated DEGs in midguts of low titer and high titer mosquitoes, and (D) biological processes of upregulated DEGs in carcasses of low titer and high titer mosquitoes. GO terms were analyzed using VectorBase.

Figure 7

KEGG pathway analysis of (A) low titer midguts, (B) low titer carcasses, (C) high titer midguts, and (D) high titer carcasses.