Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Dengue-suppressing Wolbachia strains are promising tools for arbovirus control, particularly as they have the potential to self-spread following local introductions. To test this, we followed the frequency of the transinfected Wolbachia strain wMel through Ae. aegypti in Cairns, Australia, following releases at 3 nonisolated locations within the city in early 2013. Spatial spread was analysed graphically using interpolation and by fitting a statistical model describing the position and width of the wave. For the larger 2 of the 3 releases (covering 0.97 km2 and 0.52 km2), we observed slow but steady spatial spread, at about 100–200 m per year, roughly consistent with theoretical predictions. In contrast, the smallest release (0.11 km2) produced erratic temporal and spatial dynamics, with little evidence of spread after 2 years. This is consistent with the prediction concerning fitness-decreasing Wolbachia transinfections that a minimum release area is needed to achieve stable local establishment and spread in continuous habitats. Our graphical and likelihood analyses produced broadly consistent estimates of wave speed and wave width. Spread at all sites was spatially heterogeneous, suggesting that environmental heterogeneity will affect large-scale Wolbachia transformations of urban mosquito populations. The persistence and spread of Wolbachia in release areas meeting minimum area requirements indicates the promise of successful large-scale population transformation.

BackgroundGenetic markers are widely used to understand the biology and population dynamics of disease vectors, but often markers are limited in the resolution they provide. In particular, the delineation of population structure, fine scale movement and patterns of relatedness are often obscured unless numerous markers are available. To address this issue in the major arbovirus vector, the yellow fever mosquito (Aedes aegypti), we used double digest Restriction-site Associated DNA (ddRAD) sequencing for the discovery of genome-wide single nucleotide polymorphisms (SNPs). We aimed to characterize the new SNP set and to test the resolution against previously described microsatellite markers in detecting broad and fine-scale genetic patterns in Ae. aegypti.ResultsWe developed bioinformatics tools that support the customization of restriction enzyme-based protocols for SNP discovery. We showed that our approach for RAD library construction achieves unbiased genome representation that reflects true evolutionary processes. In Ae. aegypti samples from three continents we identified more than 18,000 putative SNPs. They were widely distributed across the three Ae. aegypti chromosomes, with 47.9% found in intergenic regions and 17.8% in exons of over 2,300 genes. Pattern of their imputed effects in ORFs and UTRs were consistent with those found in a recent transcriptome study. We demonstrated that individual mosquitoes from Indonesia, Australia, Vietnam and Brazil can be assigned with a very high degree of confidence to their region of origin using a large SNP panel. We also showed that familial relatedness of samples from a 0.4 km2 area could be confidently established with a subset of SNPs.ConclusionsUsing a cost-effective customized RAD sequencing approach supported by our bioinformatics tools, we characterized over 18,000 SNPs in field samples of the dengue fever mosquito Ae. aegypti. The variants were annotated and positioned onto the three Ae. aegypti chromosomes. The new SNP set provided much greater resolution in detecting population structure and estimating fine-scale relatedness than a set of polymorphic microsatellites. RAD-based markers demonstrate great potential to advance our understanding of mosquito population processes, critical for implementing new control measures against this major disease vector.

BackgroundTraditional vector control approaches such as source reduction and insecticide spraying have limited effect on reducing Aedes aegypti population. The endosymbiont Wolbachia is pointed as a promising tool to mitigate arbovirus transmission and has been deployed worldwide. Models predict a rapid increase on the frequency of Wolbachia-positive Ae. aegypti mosquitoes in local settings, supported by cytoplasmic incompatibility (CI) and high maternal transmission rate associated with the wMelBr strain.Methodology/principle findingsWolbachia wMelBr strain was released for 20 consecutive weeks after receiving >87% approval of householders of the isolated community of Tubiacanga, Rio de Janeiro. wMelBr frequency plateued~40% during weeks 7–19, peaked 65% but dropped as releases stopped. A high (97.56%) maternal transmission was observed. Doubling releases and deploying mosquitoes with large wing length and low laboratory mortality produced no detectable effects on invasion trend. By investigating the lab colony maintenance procedures backwardly, pyrethroid resistant genotypes in wMelBr decreased from 68% to 3.5% after 17 generations. Therefore, we initially released susceptible mosquitoes in a local population highly resistant to pyrethroids which, associated with the over use of insecticides by householders, ended jeopardizing Wolbachia invasion. A new strain (wMelRio) was produced after backcrossing wMelBr females with males from field to introduce mostly pyrethroid resistance alleles. The new strain increased mosquito survival but produced relevant negative effects on Ae. aegypti fecundity traits, reducing egg clutche size and egg hatch. Despite the cost on fitness, wMelRio successful established where wMelBr failed, revealing that matching the local population genetics, especially insecticide resistance background, is critical to achieve invasion.Conclusions/significanceLocal householders support was constantly high, reaching 90% backing on the second release (wMelRio strain). Notwithstanding the drought summer, the harsh temperature recorded (daily average above 30°C) did not seem to affect the expression of maternal transmission of wMel on a Brazilian background. Wolbachia deployment should match the insecticide resistance profile of the wild population to achieve invasion. Considering pyrethroid-resistance is a widely distributed phenotype in natural Ae. aegypti populations, future Wolbachia deployments must pay special attention in maintaining insecticide resistance in lab colonies for releases.

Wolbachia are endosymbionts found in many insects with the potential to suppress vectorborne diseases, particularly through interfering with pathogen transmission. Wolbachia strains are highly variable in their effects on hosts, raising the issue of which attributes should be selected to ensure that the best strains are developed for disease control. This depends on their ability to suppress viral transmission, invade host populations, persist without loss of viral suppression and not interfere with other control strategies. The potential to achieve these objectives is likely to involve evolutionary constraints; viral suppression may be limited by the ability of infections to spread due to deleterious host fitness effects. However, there are exceptions to these patterns in both natural infections and in novel associations generated following interspecific transfer, suggesting that pathogen blockage, deleterious fitness effects and changes to reproductive biology might be at least partly decoupled to achieve ideal infection attributes. The stability of introduced Wolbachia and its effects on viral transmission remain unclear, but rapid evolutionary changes seem unlikely. Although deliberate transfers of Wolbachia across species remain particularly challenging, the availability of strains with desirable attributes should be expanded, taking advantage of the diversity available across thousands of strains in natural populations.

The endosymbiotic bacterium Wolbachia suppresses the capacity for arbovirus transmission in the mosquito Aedes aegypti, and can spread spatially through wild mosquito populations following local introductions. Recent introductions in Cairns, Australia have demonstrated slower than expected spatial spread. Potential reasons for this include: (i) barriers to Ae. aegypti dispersal; (ii) higher incidence of long-range dispersal; and (iii) intergenerational loss of Wolbachia. We investigated these three potential factors using genome-wide single-nucleotide polymorphisms (SNPs) and an assay for the Wolbachia infection wMel in 161 Ae. aegypti collected from Cairns in 2015. We detected a small but significant barrier effect of Cairns highways on Ae. aegypti dispersal using distance-based redundancy analysis and patch-based simulation analysis. We detected a pair of putative full-siblings in ovitraps 1312 m apart, indicating long-distance female movement likely mediated by human transport. We also found a pair of full-siblings of different infection status, indicating intergenerational loss of Wolbachia in the field. These three factors are all expected to contribute to the slow spread of Wolbachia through Ae. aegypti populations, though from our results it is unclear whether Wolbachia loss and long-distance movement are sufficiently common to reduce the speed of spatial spread appreciably. Our findings inform the strategic deployment of Wolbachia-infected mosquitoes during releases, and show how parameter estimates from laboratory studies may differ from those estimated using field data. Our landscape genomics approach can be extended to other host/symbiont systems that are being considered for biocontrol.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the “Debug” Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Medically important arboviruses such as dengue, Zika, and chikungunya viruses are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that transmission can be influenced by mosquito viromes. Herein RNA-Seq was used to characterize RNA metaviromes of wild-caught Ae. aegypti from Bangkok (Thailand) and from Cairns (Australia). The two mosquito populations showed a high degree of similarity in their viromes. BLAST searches of assembled contigs suggest up to 27 insect-specific viruses may infect Ae. aegypti, with up to 23 of these currently uncharacterized and up to 16 infecting mosquitoes from both Cairns and Bangkok. Three characterized viruses dominated, Phasi Charoen-like virus, Humaita-Tubiacanga virus and Cell fusing agent virus, and comparisons with other available RNA-Seq datasets suggested infection levels with these viruses may vary in laboratory-reared mosquitoes. As expected, mosquitoes from Bangkok showed higher mitochondrial diversity and carried alleles associated with knock-down resistance to pyrethroids. Blood meal reads primarily mapped to human genes, with a small number also showing homology with rat/mouse and dog genes. These results highlight the wide spectrum of data that can be obtained from such RNA-Seq analyses, and suggests differing viromes may need to be considered in arbovirus vector competence studies.