RNA sequencing analysis of viromes of Aedes albopictus and Aedes vexans collected from NEON sites

Abstract

Climate change is significantly impacting the geographic range of many animal species and their associated microorganisms, hence influencing emergence of vector-borne diseases. Mosquito-borne viruses represent a potential major reservoir of human pathogens, highlighting the need for improved understanding of ecological factors associated with variation in the mosquito viral community (virome). Here, a subtractive hybridization method coupled with RNAseq of individual mosquito specimens was used to profile RNA viromes of individual co-occurring Aedes albopictus and Aedes vexans mosquitoes across a 2,000 km spatial scale. Samples were collected and archived by the National Ecological Observatory Network (NEON) from four ecologically variable sites in the Southeastern United States between 2018 and 2019. Results of multivariate analysis suggest that mosquito species are an important factor in RNA viral community composition. Significantly higher viral diversity was detected in A. albopictus compared to A.vexans. However, season, year, and site of sample collection did not show strong association with virome profiles, supporting the hypothesis that factors unique to the mosquito host species (e.g., larval habitat or vector competence) influence the structure of mosquito viromes.

Keywords: Aedes albopictus; Aedes vexans; arbovirus; mosquito; sequencing.

Fig. 1.

Map of study area showing the four NEON sites from which mosquitoes were collected during 2018–2019. Larger circles indicate “core” sites sampled every two weeks during the active mosquito season, while smaller circles indicate “gradient” sites sampled every four weeks. Due to lack of sufficient samples of Aedes vexans collected at SERC in 2018, a total of three individuals were sequenced from the nearby SCBI site. NEON is a national observatory with ecological sampling occurring at 47 terrestrial and 34 aquatic sites spread across 20 ecoclimatic domains. Blue lines delineate the ecodomain boundaries, and sites without circles are areas where mosquitoes are collected by NEON but with fewer replicate samples for both species to include in this project.

Fig. 2.

Relative abundance of reads for virus families within each sampling category for (A) the RVDB dataset and (B) the Lazypipe dataset. Temporal Category indicates year followed by “.E” for early season or “.L” for late season samples. The average depth of sequencing for the CLBJ, ORNL, SCBI, SERC, and TALL samples were approximately 115 million, 107 million, 126 million, 124 million, and 110 million reads, respectively.

Fig. 3.

Sankey diagram depicting viruses detected within each vector as percent of total vector–virus pairs (n = 373) present using the RVDB dataset.

Fig. 4.

Heatmap showing proportion of mosquitoes and virus species in each sample group using the RVDB dataset. Sample Group indicates the year followed by “.E” for early season or “.L” for late season samples.

Fig. 5.

NMDS plots of mosquito viromes using the RVDB dataset grouped by (A) spatiotemporal category and species, where species are depicted with filled or open symbols, site with color, and time with differently shaped symbols. Additional diagrams show the same data by (B) species, (C) site, (D) time of season, and (E) year. Ellipses indicate 95% CI. The stress value is 0.06.