Deciphering the Tissue Tropism of the RNA Viromes Harbored by Field-Collected Anopheles sinensis and Culex quinquefasciatus

Abstract

Arboviruses and insect-specific viruses (ISVs) are two major types of viruses harbored by mosquitoes that are distinguished by the involvement of vertebrate hosts in their transmission cycles. While intensive studies have focused on the transmission, tissue tropism, and evolution of arboviruses, these characteristics are poorly investigated in ISVs, which dominate the mosquito virome. Therefore, in this study, we collected two mosquito species, Anopheles sinensis and Culex quinquefasciatus, in the field and used a metatranscriptomics approach to characterize their RNA viromes in different tissues, such as the midgut, legs, salivary gland, eggs, and the remainder of the carcass. Blood-engorged individuals of these species were captured in 3 locations, and 60 mosquitoes were pooled from each species and location. A total of 40 viral species from diverse viral taxa associated with all viral RNA genome types were identified, among which 19 were newly identified in this study. According to the current viral taxonomy, some of these viruses, such as Yancheng Anopheles associated virus 2 (Narnaviridae) and Jiangsu Anopheles-related virus (Ghabrivirales), were novel. The two investigated mosquito species generally harbored distinct viromes. Nevertheless, the viruses were generally shared among different tissue types to various degrees. Specifically, the eggs possessed a viral community with significantly lower diversity and abundance than those in other tissues, whereas the legs and salivary glands exhibited higher viral abundance. The compositions and distributions of the viromes of different mosquito tissues were demonstrated for the first time in our study, providing important insight into the virome dynamics within individual mosquitoes. IMPORTANCE ISVs are considered to be ancestral to arboviruses. Because of their medical importance, arboviruses have been well studied from the aspects of their transmission mode, evolution of dual-host tropism, and genetic dynamics within mosquito vectors. However, the mode of ISV maintenance is poorly understood, even though many novel ISVs have been identified with the emergence of sequencing technology. In our study, in addition to the identification of a diverse virus community, the tissue tropism of RNA viromes harbored by two field-collected mosquito species was demonstrated for the first time. According to the results, the virus communities of different tissues, such as the salivary glands, midguts, legs, and eggs, can help us understand the evolution, transmission routes, and maintenance modes of mosquito-specific viruses in nature.

Keywords: Anopheles sinensis; Culex quinquefasciatus; insect-specific viruses; tissue tropism; virome.

FIG 1

Overview of the sequencing data and composition. (a) Proportions of assembled contigs from different superkingdoms. “Dark matter” refers to the sequences that were unclassified, including a tiny proportion of sequences from Archaea. (b) Histogram displaying the read number of viruses and the host RPL8 gene in different pools. The dotted line indicates the reads per million reads (RPM) values of the viromes in different pools. The pools are named as follows. The first letter indicates the species (A, An. sinensis; C, Cx. quinquefasciatus), the second letter indicates the tissue type (M, midgut; L, leg; S, salivary gland; E, egg; C, the remainder of the carcass), and the number indicates the location (1, Yancheng; 2, Liyang). All of the Cx. quinquefasciatus mosquitoes were from Yixing.

FIG 2

Comparison and connectivity of the viromes in the tissue pools of An. sinensis and Cx. quinquefasciatus. (a) Alpha diversity of viruses in the two mosquito species at the viral species level; (b) nonmetric multidimensional scaling (NMDS) of viruses at the viral species level; (c) Venn diagram presenting the relationships among the virome compositions of mosquitoes at three locations; (d to f): Venn diagrams presenting the relationships of the virome compositions among various tissue pools of An. sinensis from locations 1 and 2 and Cx. quinquefasciatus. The histograms below the Venn diagrams represent the number of viral species in various mosquito species or tissue pools.

FIG 3

RPM values of each virus in different tissue pools. The data are displayed as the average values for An. sinensis from the two locations.

FIG 4

The heat map displays the RPM values of each virus in different tissue pools under the log₂ function. In the pool names, the first letter indicates the species (A, An. sinensis; C, Cx. quinquefasciatus), the second letter indicates the tissue type (M, midgut; L, leg; S, salivary gland; E, egg; C, the remainder of the carcass), and the number indicates the location (1, Yancheng; 2, Liyang).

FIG 5

Box plot of the viral read number dynamics of dominant viral species among different mosquito tissue pools. Each colored dot indicates the viral reads normalized using the host RPL8 gene under the log₂ function in different tissue pools. To reveal the dynamic trend with a visualized range, the viral read/RPSL8 ratio was adjusted by adding 1. Only the dominant viral species were considered. The box indicates the 25 to 75% range of normalized viral reads of each pool. The white line within the box indicates the median, and the white square indicates the mean value. Panels a, b, and c present the results for mosquitoes from Yancheng, Liyang, and Yixing, respectively.

FIG 6

Phylogeny of the detected RNA viruses with related references in different taxonomic groups. The annotation is as follows. In the dots and viral names, orange indicates the viruses detected in An. sinensis, and green indicates those in Cx. quinquefasciatus. Due to the incongruous alignment resulting from some partial sequences, some phylogenetic trees of the same taxonomic status were constructed separately for the coordinated topology (such as Hepe-like, Chu-like, and Rhabdo-like).