Establishment of Culex modestus in Belgium and a Glance into the Virome of Belgian Mosquito Species

Abstract

Culex modestus mosquitoes are considered potential transmission vectors of West Nile virus and Usutu virus. Their presence has been reported across several European countries, including one larva detected in Belgium in 2018. In this study, mosquitoes were collected in the city of Leuven and surrounding areas in the summers of 2019 and 2020. Species identification was performed based on morphological features and partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The 107 mosquitoes collected in 2019 belonged to eight mosquito species, Culex pipiens (24.3%), Cx. modestus (48.6%), Cx. torrentium (0.9%), Culiseta annulata (0.9%), Culiseta morsitans (0.9%), Aedes sticticus (14.0%), Aedes cinereus (9.3%), and Anopheles plumbeus (0.9%), suggesting the presence of an established Cx. modestus population in Belgium. The collection of Cx. modestus mosquitoes at the same locations in 2020 confirmed their establishment in the region. Haplotype network analysis of the COI sequences for Cx. modestus showed that the Belgian population is rather diverse, suggesting that it may have been established in Belgium for some time. The Belgian Cx. modestus population was most closely related to populations from the United Kingdom and Germany. Characterization of the virome of the collected mosquitoes resulted in the identification of at least 33 eukaryotic viral species. Nine (nearly) complete genomes belonging to 6 viral species were identified, all of which were closely related to known viruses. In conclusion, here, we report the presence of Cx. modestus in the surrounding areas of Leuven, Belgium. As this species is considered to be a vector of several arboviruses, the implementation of vector surveillance programs to monitor this species is recommended.IMPORTANCECulex modestus mosquitoes are considered to be a potential "bridge" vector, being able to transmit pathogens between birds as well as from birds to mammals, including humans. In Belgium, this mosquito species was considered absent until the finding of one larva in 2018 and subsequent evidence of a large population in 2019 to 2020 described here. We collected mosquitoes in the summers of 2019 and 2020 in the city of Leuven and surrounding areas. The mosquito species was identified by morphological and molecular methods, demonstrating the presence of Cx. modestus in this region. The ability of mosquitoes to transmit pathogens can depend on several factors, one of them being their natural virus composition. Therefore, we identified the mosquito-specific viruses harbored by Belgian mosquitoes. As Cx. modestus is able to transmit viruses such as West Nile virus and Usutu virus, the establishment of this mosquito species may increase the risk of virus transmission in the region. It is thus advisable to implement mosquito surveillance programs to monitor this species.

Keywords: Belgium; Culex modestus; haplotype; mosquito; virome.

FIG 1

Mosquito species collected in Leuven, Belgium, in 2019. (A) Distribution of mosquito species captured during the summer of 2019 across all locations sampled in Leuven. (B) Distribution of mosquito species across habitat types in Leuven. Mosquito species are marked in different colors. The number of specimens is indicated in the bar chart.

FIG 2

ML tree of the COI sequences of 21 culicid species. Sequences derived from mosquitoes collected in Leuven are collapsed with the reference sequences for Cx. modestus. The collapsed branch is expanded in the panel on the right. GenBank accession numbers are in parentheses.

FIG 3

Median-joining network constructed with 228 COI sequences of Cx. modestus from 9 countries in Europe. Each circle represents a haplotype. The size of the circle corresponds to the number of specimens sharing that specific haplotype. Each country is represented by a color, described in the key. Mosquito collections in Belgium are separated per year to visualize the allocation of haplotypes in the network. The gray backgrounds represent both lineages found and the distinction of these two groups.

FIG 4

Summary information and viral composition of sequenced samples. (A) Location, mosquito species, and number of specimens present in each of the sequenced pools. (B) Bar plots representing the abundance of reads belonging to distinct viral families per pool. The number of eukaryotic viral reads per pool is given on top of each bar.

FIG 5

Heat map of normalized read counts for eukaryotic viruses. The heat map shows the normalized count on a log₂ scale of reads mapping to the assembled contigs of each eukaryotic virus. Next to the taxonomic annotation, obtained by DIAMOND and KronaTools, the average BLASTx identity for all contigs representing a viral species is depicted by the shaded blue boxes. Hierarchical clustering of the columns is based on the Bray-Curtis distance matrix calculated from the normalized read counts.

FIG 6

(Nearly) complete meta-assembled genomes identified in mosquitoes collected during the summer of 2019. Bootstrap support values are shown next to the nodes. Complete MAGs are colored in red. (A) Midpoint-rooted ML tree of all complete genomes related to Culex inatomii totivirus, selected after BLASTn analysis. (B) Midpoint-rooted ML tree of all Mesoniviridae family members. (C) Midpoint-rooted ML tree of all complete genomes related to our Yongsan iflavirus 1 and Culex iflavi-like virus 4 genomes, selected after BLASTn analysis. (D) ML tree of all complete genomes related to Yongsan negev-like virus 1, selected after BLASTn analysis. Negevirus was used as the outgroup. (E) Midpoint-rooted ML tree of all complete genomes related to the recovered Riverside virus 1. GenBank accession numbers are in parentheses.