A total of 2222 mosquitoes were collected and identified to species level through morphological identification under a stereomicroscope. They were classified into four species: An. sinensis, Ar. subalbatus, Cx. quinquefasciatus, and Cx. tritaeniorhynchus, with 387, 852, 745, and 238 mosquitoes from each species, respectively. The four species are very common in the Yichang region, Hubei, and the dominant species in 2014 were Ar. subalbatus (38.3%) followed by Cx. quinquefasciatus (33.5%).
A total of 6, 535, 325, 7, 183, 603, 6, 714, 707 and 8, 909, 955 clean, paired reads and 251, 198, 284, and 186 assembled contigs were generated from An. sinensis, Ar. subalbatus, Cx. quinquefasciatus, and Cx. tritaeniorhynchus, respectively, through Illumina Miseq sequencing. After querying of the RefSeq Complete Genomes database through the Kaiju Web Server, the result indicated that there were many reads that corresponded to known viral sequences (Table 1). The percentage of the reads that matched to viral sequences were 52.5%, 30.9%, 85.3%, and 71.1% in An. sinensis, Ar. subalbatus, Cx. quinquefasciatus, and Cx. tritaeniorhynchus respectively, and highest prevalence of virus associated reads were observed in Cx. quinquefasciatus.
Table 1. Reads/contigs in each mosquito species.
Taxonomic classification was conducted based on viral families. The results indicate that the virus-related reads present in the four mosquito viromes are distributed across 26 virus families, together with an unclassified group (Supplementary Table S1).
Anelloviridae, Circoviridae, Genomoviridae, Iridoviridae, Mesoniviridae, Microviridae, Myoviridae, Parvoviridae, Phenuiviridae, and Podoviridae were the top ten significantly different viral families (Fig. 1). According to the detailed analysis results in Supplementary Table S1, many reads related to Iridoviridae, Poxviridae, Totiviridae, and Peribunyaviridae were found in An. sinensis, while they were rarely detected in the other three species. In addition, the reads that matched to Phenuiviridae and Siphoviridae were identified in Ar. subalbatus, however rarely found in the other species.
Figure 1. Top ten significantly different viral families among four mosquito species. Frequency is the rate of the number of reads belong to a viral family to the total number of viral reads in one mosquito sample. The frequencies of Anelloviridae and Parvoviridae in four samples dramatically fluctuated.
To detect differences in virome structures among the different mosquito species, a taxonomic heatmap and hierarchical cluster analysis were conducted based on a distance matrix (Fig. 2). The heatmap analysis based on virus family showed that each species had a special virome structure. For instance, in An. sinensis mosquitoes, Parvoviridae, Reoviridae, Poxviridae, Iridoviridae, Anelloviridae, Totiviridae, Myoviridae, Inoviridae, and Peribunyaviridae were found to be present in abundant quantities while in Ar. subalbatus, Iflaviridae, Microviridae, Siphoviridae, Circoviridae, and Phenuiviridae were the most abundant virus families. However, the abundant viral families in Cx. quinquefasciatus species were found to be Genomoviridae, Ascoviridae, Tymoviridae, Marseilleviridae, and Polydnaviridae. In Cx. tritaeriorhynchus mosquitoes, Mesoniviridae and Geminiviridae were detected in abundant quantities. The cluster results showed Cx. quinquefasciatus and Cx. tritaeniorhynchus grouped together, which indicated the correlation of the virome structures in these two species were closer than they were to the other species, to some extent.For example, the Phycodnaviridae, Mimiviridae and Rhabdoviridae viral families were found to be in abundant or moderate levels in both Culex species.
Figure 2. Heatmap based on 26 viral families. Heatmap was constructed based on the distance matrix, which was calculated by Euclidean distance method according to the number of reads belonging to each viral family in four mosquito species. The X axis represents the sample names of mosquito species, the Y axis represents the names of viral families. The color red to blue stands for the highest to lowest abundant of viral reads in each species according to viral family.
The information for contigs that matched to the known viruses in each sample is summarized in Table 2. In An. sinensis, three types of Torque teno sus virus (1a, 1b, and k2) were detected, however, none of them were detected in Cx. tritaeniorhynchus. Three types of mesonivirus were found in Cx. tritaeniorhynchus, and two types in Ar. subalbatus, but none of them were detected in An. sinensis or Cx. quinquefasciatus. The Wuhan fly virus was only detected in An. sinensis. BAV was found in An. sinensis and Cx. tritaeniorhynchus. Large amount of contigs matched to Cx. tritaeniorhynchus rhabdovirus, Wenzhou sobemo-like virus 3, and Wuhan mosquito virus 8 in the two Culex species. Hubei mosquito virus 2 was found in all four species. Many newly identified viruses, such as the Hubei mosquito virus, Hubei tombus-like virus, Wenzhou sobemo-like, and Zhejiang mosquito virus reported by Shi et al. (2016), were detected in our mosquito samples (Table 2).
Table 2. Information on contig hits to the virus database in four mosquito species.
In addition, several viral sequences which maybe belong to novel viruses (low identity to known virus) for the first time presented in mosquitoes were discovered (Table 2). For example, the Tanay virus was only previously reported in Culex spp. or Armigeres spp. in the Philippines (Li et al. 2014). However, there was one contig matched with Tanay virus (with 46% identity) in our An. sinensis sample. Several contigs hit to Wuhan fly virus 1, Wuhan ant virus, Beihai mantis shrimp virus 6, Hubei picorna-like virus 41, Hubei rhabdo-like virus 1, Sanxia water strider virus 8, or Wuhan house centipede virus 6, with the identity ranging from 29% to 76%, which was the first time that these viruses have been found in mosquitoes. In future work, we will try to isolate them and conduct further analysis.