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. 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. 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.

Domesticating Zika virus: Why hasn't Zika virus (ZIKV) disease caused as much devastation in Africa, its continent of origin, as it has in the Americas? Outside of Africa, this flavivirus is transmitted by a ubiquitous mosquito subspecies, Aedes aegypti aegypti , which emerged from the African forerunner subspecies A. aegypti formosus and acquired a preference for human blood and a peridomestic lifestyle. Now, this subspecies colonizes many intertropical cities, aided by climate change and human trash. Aubry et al. tested 14 laboratory mosquito colonies for their relative susceptibility to ZIKV. Quantitative trait locus mapping showed differences on chromosome 2 between mosquitoes from Gabon and Guadeloupe. Mouse infection experiments revealed that African mosquitoes transmitted a smaller virus inoculum than the South American insects. Increased susceptibility coupled with the ability of A. aegypti aegypti to breed in any discarded object containing water has amplified the problematic nature of this virus as it has circumnavigated the world. Science , this issue p. 991

In the Guadeloupe and Saint Martin islands, Aedes aegypti mosquitoes are the only recognized vectors of dengue, chikungunya, and Zika viruses. For around 40 years, malathion was used as a mosquito adulticide and temephos as a larvicide. Since the European Union banned the use of these two insecticide molecules in the first decade of the 21st century, deltamethrin and Bacillus thuringiensis var. israelensis are the remaining adulticide and larvicide, respectively, used in Guadeloupe. In order to improve the management of vector control activities in Guadeloupe and Saint Martin, we investigated Ae. aegypti resistance to and mechanisms associated with deltamethrin, malathion, and temephos. Ae. aegypti mosquitoes were collected from six different localities of Guadeloupe and Saint Martin. Larvae were used for malathion and temephos bioassays, and adult mosquitoes for deltamethrin bioassays, following World Health Organization recommendations. Knockdown resistance (Kdr) genotyping for V1016I and F1534C mutations, and expression levels of eight enzymes involved in detoxification mechanisms were examined in comparison with the susceptible reference Bora Bora strain. Resistance ratios (RR50) calculated for Ae. aegypti larvae showed high resistance levels to temephos (from 8.9 to 33.1-fold) and low resistance levels to malathion (from 1.7 to 4.4-fold). Adult females displayed moderate resistance levels to deltamethrin regarding the time necessary to affect 50% of individuals, varying from 8.0 to 28.1-fold. Molecular investigations on adult mosquitoes showed high resistant allele frequencies for V1016I and F1534C (from 85 to 96% and from 90 to 98%, respectively), as well as an overexpression of the glutathione S-transferase gene, GSTe2, the carboxylesterase CCEae3a, and the cytochrome genes 014614, CYP6BB2, CYP6M11, and CYP9J23. Ae. aegypti populations from Guadeloupe and Saint Martin exhibit multiple resistance to organophosphates (temephos and malathion), and pyrethroids (deltamethrin). The mechanisms associated with these resistance patterns show strong frequencies of F1534C and V1016I Kdr mutations, and an over-expression of CCEae3a, GSTe2, and four cytochrome P450 genes (014614, CYP9J23, CYP6M11, CYP6BB2). These results will form the baseline for a deeper understanding of the insecticide resistance levels and associated mechanisms of Ae. aegypti populations and will be used to improve vector control strategies in Guadeloupe and Saint Martin.

Host-associated microbes, collectively known as the microbiota, play an important role in the biology of multicellular organisms. In mosquito vectors of human pathogens, the gut bacterial microbiota influences vectorial capacity and has become the subject of intense study. In laboratory studies of vector biology, genetic effects are often inferred from differences between geographically and genetically diverse colonies of mosquitoes that are reared in the same insectary. It is unclear, however, to what extent genetic effects can be confounded by uncontrolled differences in the microbiota composition among mosquito colonies. To address this question, we used 16S metagenomics to compare the midgut bacterial microbiome of six laboratory colonies of Aedes aegypti recently derived from wild populations representing the geographical range and genetic diversity of the species. We found that the diversity, abundance, and community structure of the midgut bacterial microbiome was remarkably similar among the six different colonies of Ae. aegypti, regardless of their geographical origin. We also confirmed the relatively low complexity of bacterial communities inhabiting the mosquito midgut. Our finding that geographically diverse colonies of Ae. aegypti reared in the same insectary harbor a similar gut bacterial microbiome supports the conclusion that the gut microbiota of adult mosquitoes is environmentally determined regardless of the host genotype. Thus, uncontrolled differences in microbiota composition are unlikely to represent a significant confounding factor in genetic studies of vector biology.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology (MALDI-TOF MS) is an innovative tool that has been shown to be effective for the identification of numerous arthropod groups including mosquitoes. A critical step in the implementation of MALDI-TOF MS identification is the creation of spectra databases (DB) for the species of interest. Mosquito legs were the body part most frequently used to create identification DB. However, legs are one of the most fragile mosquito compartments, which can put identification at risk. Here, we assessed whether mosquito thoraxes could also be used as a relevant body part for mosquito species identification using a MALDI-TOF MS biotyping strategy; we propose a double DB query strategy to reinforce identification success. Thoraxes and legs from 91 mosquito specimens belonging to seven mosquito species collected in six localities from Guadeloupe, and two laboratory strains, Aedes aegypti BORA and Aedes albopictus Marseille, were dissected and analyzed by MALDI-TOF MS. Molecular identification using cox1 gene sequencing was also conducted on representative specimens to confirm their identification. MS profiles obtained with both thoraxes and legs were highly compartment-specific, species-specific and species-reproducible, allowing high identification scores (log-score values, LSVs) when queried against the in-house MS reference spectra DB (thorax LSVs range: 2.260–2.783, leg LSVs range: 2.132–2.753). Both thoraxes and legs could be used for a double DB query in order to reinforce the success and accuracy of MALDI-TOF MS identification.