Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Aug 28;7(1):121.
doi: 10.1186/s40168-019-0734-2.

Stable distinct core eukaryotic viromes in different mosquito species from Guadeloupe, using single mosquito viral metagenomics

Chenyan Shi  1 , Leen Beller  1 , Ward Deboutte  1 , Kwe Claude Yinda  1   2 , Leen Delang  3 , Anubis Vega-Rúa  4 , Anna-Bella Failloux  5 , Jelle Matthijnssens  6
Affiliations

Stable distinct core eukaryotic viromes in different mosquito species from Guadeloupe, using single mosquito viral metagenomics

Chenyan Shi et al. Microbiome. .

Abstract

Background: Mosquitoes are the most important invertebrate viral vectors in humans and harbor a high diversity of understudied viruses, which has been shown in many mosquito virome studies in recent years. These studies generally performed metagenomics sequencing on pools of mosquitoes, without assessment of the viral diversity in individual mosquitoes. To address this issue, we applied our optimized viral metagenomics protocol (NetoVIR) to compare the virome of single and pooled Aedes aegypti and Culex quinquefasciatus mosquitoes collected from different locations in Guadeloupe, in 2016 and 2017.

Results: The total read number and viral reads proportion of samples containing a single mosquito have no significant difference compared with those of pools containing five mosquitoes, which proved the feasibility of using single mosquito for viral metagenomics. A comparative analysis of the virome revealed a higher abundance and more diverse eukaryotic virome in Aedes aegypti, whereas Culex quinquefasciatus harbors a richer and more diverse phageome. The majority of the identified eukaryotic viruses were mosquito-species specific. We further characterized the genomes of 11 novel eukaryotic viruses. Furthermore, qRT-PCR analyses of the six most abundant eukaryotic viruses indicated that the majority of individual mosquitoes were infected by several of the selected viruses with viral genome copies per mosquito ranging from 267 to 1.01 × 108 (median 7.5 × 106) for Ae. aegypti and 192 to 8.69 × 106 (median 4.87 × 104) for Cx. quinquefasciatus. Additionally, in Cx. quinquefasciatus, a number of phage contigs co-occurred with several marker genes of Wolbachia sp. strain wPip.

Conclusions: We firstly demonstrate the feasibility to use single mosquito for viral metagenomics, which can provide much more precise virome profiles of mosquito populations. Interspecific comparisons show striking differences in abundance and diversity between the viromes of Ae. aegypti and Cx. quinquefasciatus. Those two mosquito species seem to have their own relatively stable "core eukaryotic virome", which might have important implications for the competence to transmit important medically relevant arboviruses. The presence of Wolbachia in Cx. quinquefasciatus might explain (1) the lower overall viral load compared to Ae. aegypti, (2) the identification of multiple unknown phage contigs, and (3) the difference in competence for important human pathogens. How these viruses, phages, and bacteria influence the physiology and vector competence of mosquito hosts warrants further research.

Keywords: Aedes aegypti; Core virome; Culex quinquefasciatus; Eukaryotic virome; Guadeloupe; Phageome; Single mosquito; Viral metagenomics.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Comparison between NGS reads of single mosquito and pooled mosquitoes. a Proportion of each taxonomic category in single mosquito and pooled mosquitoes based on reads number. Legend contains the percentage of each category, as well as the p values of Wilcoxon test on the proportion of each category between single mosquito and pooled mosquitoes. b Comparison of total reads number mapped to the nr contigs collection in single mosquito and pooled mosquitoes. The nr contigs collection were obtained by removing the redundancy at 95% nucleotide identity over 80% of the length from all the de novo assembled contigs (> 500 bp) of all 36 samples. c Comparison of viral reads proportion (eukaryotic viruses, phages and unassigned virus) in single mosquito and pooled mosquitoes
Fig. 2
Fig. 2
Comparison between viral reads in Aedes aegypti and Culex quinquefasciatus per sample/pool. a Proportion of eukaryotic virus, bacteriophage, bacteriophageTBC, and unassigned virus in each sample/pool, for Aedes aegypti and Culex quinquefasciatus. The samples are ranked in a descending proportion of eukaryotic virus reads. The samples marked with red dots are pools containing five mosquitoes, whereas the other samples contain individual mosquitoes. Samples Ab-AAF-1-3 is labeled with a star symbol. b Comparison of the proportion of eukaryotic virus reads in the two mosquito species. c Comparison of the proportion of bacteriophage reads in the two mosquito species
Fig. 3
Fig. 3
Alpha and beta diversity of the virome in Aedes aegypti and Culex quinquefasciatus samples/pools. a Alpha diversity of eukaryotic viruses in Aedes aegypti and Culex quinquefasciatus on vOTU and species level. b Alpha diversity of bacteriophage contigs in Aedes aegypti and Culex quinquefasciatus on vOUT level. Pairwise ANOVA: p < 0.01 (*), p < 0.001 (**), p < 0.0001 (***). c Non-metric multi-dimensional scaling (NMDS) of eukaryotic viruses on viral species level. Samples Ab-AAF-1-3 is labeled with text and a star symbol. STRESS = 0.0425, PERMANOVA test on mosquito species: p = 0.001, R2 = 0.126. d NMDS of bacteriophages on vOTU level. Samples Ab-AAF-1-3 is labeled with text and a star symbol. STRESS = 0.034, PERMANOVA test on mosquito species: p = 0.001, R2 = 0.311
Fig. 4
Fig. 4
Normalized abundance of eukaryotic viral species. The heatmap shows the normalized reads counts by metagenomeSeq on log2 scale. The hierarchical clustering is based on the Euclidean distance matrix calculated from the normalized reads count. The viral species names shown in the heatmap are from the taxonomic annotation by DIAMOND and KronaTools. For each of the contigs assigned to a particular species, the ORF with the highest BLASTx identity to a reference sequence was taken, and the average identity of these different ORFs is shown in the shaded blue boxes. The red-shaded viruses were selected for qRT-PCR analysis and the names of novel viruses are shown between brackets. The samples marked with red dots are pools containing five mosquitoes and the one with star is the special sample Ab-AAF-1-3
Fig. 5
Fig. 5
Phylogenetic trees of selected eukaryotic viruses identified in 2016 and 2017 samples. a ML phylogeny of Luteoviridae and Sobemovirus-related viruses based on amino acid sequence of RdRp. b ML phylogeny of Phasivirus-related viruses based on amino acid sequence of RdRp. c ML phylogeny of Totiviridae-related viruses based on amino acid sequence of RdRp. d ML phylogeny of Mononegavirales-related viruses based on amino acid sequence of RdRp. e ML phylogeny of Quaranjavirus-related viruses based on the amino acid sequence of PB1. f ML phylogeny of Rhabdoviridae-related viruses based on amino acid sequence of RdRp. g ML phylogeny of Tymoviridae-related viruses based on amino acid sequence of RdRp. The most closely related references are in blue. Viruses identified from Aedes aegypti in 2016 and 2017 are orange and red, respectively. Viruses identified from the unique sample Ab-AAF-1-3 are marked with a gray triangle. Viruses identified from Culex quinquefasciatus in 2016 and 2017 are in light green and dark green, respectively
Fig. 6
Fig. 6
Quantification of GMV, PCPLV, AANV, GAATV, GCLTV, and GCRV in mosquito populations. a Copy number of each screened virus in individual Aedes aegypti or Culex quinquefasciatus. Y-axis is in log scale. The red lines indicate the ten copies, which was used as threshold to calculate the positive rate. The NGS positive rates are calculated from the reads abundance, using one read as threshold. b Total viral genome copies in each individual mosquito. The light orange and green bars indicate the total viral genome copies per individual of Aedes aegypti and Culex quinquefasciatus, respectively. Six different symbols with difference colors indicate the genome copies of each detected viruses
Fig. 7
Fig. 7
Marker genes identification and correlation analysis. a Heatmap of detected marker genes (cox1, gyrB, and recA) in NGS data of Aedes aegypti and Culex quinquefasciatus pools. The color of the heat map indicates the RPKM of the genes. The Aedes aegypti and Culex quinquefasciatus pools are highlighted with orange and green background, respectively. Pools containing five mosquitoes are marked with red dots and the sample marked with a star is the special sample Ab-AAF-1-3. b Correlation analysis on relative abundance of confirmed phage contigs (> 1500 bp), bacteria, and mosquito marker genes. The gradation of red color in the circle indicates the degree of positive correlation. The bigger size of the circle associates with lower p value. Only the correlations with an adjusted p value less than 0.01 are shown in the figure. The marker genes are labeled with red font color and phage contigs are labeled with black font color. Phage contigs of which WIsH predicted the genus Wolbachia as the host (p < 0.001) are marked in gray
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. French guiana, Guadeloupe, and Martinique. Health in the Americas, 2012 Edition: Country Volume.
    1. Larrieu S, et al. Dengue outbreaks: a constant risk for Reunion Island. Results from a seroprevalence study among blood donors. Trans R Soc Trop Med Hyg. 2014;108(1):57–59. doi: 10.1093/trstmh/trt110. - DOI - PubMed
    1. Quénel P, et al. Contributions de la recherche virologique, clinique, épidémiologique, socio comportementale et en modélisation mathématique au contrôle de la dengue dans les DFA. Bulletin de veille sanitaire. 2009;3:1–16.
    1. INVS (Institut de Veille Sanitaire), S.p.F., Le chikungunya dans les Antilles Bulletin du 17 novembre au 14 décembre 2014. le point épidémio N°34/2014 2014: p. 1–6.
    1. Van Bortel W, et al. Chikungunya outbreak in the Caribbean region, December 2013 to March 2014, and the significance for Europe. Euro Surveill. 2014;19(13). - PubMed

Publication types

LinkOut - more resources