Improved reference genome of Aedes aegypti informs arbovirus vector control.

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.

Genetic evidence for the origin of Aedes aegypti, the yellow fever mosquito, in the southwestern Indian Ocean.

Aedes aegypti is among the best-studied mosquitoes due to its critical role as a vector of human pathogens and ease of laboratory rearing. Until now, this species was thought to have originated in continental Africa, and subsequently colonized much of the world following the establishment of global trade routes. However, populations of this mosquito on the islands in the southwestern Indian Ocean (SWIO), where the species occurs with its nearest relatives referred to as the Aegypti Group, have received little study. We re-evaluated the evolutionary history of Ae. aegypti and these relatives, using three data sets: nucleotide sequence data, 18,489 SNPs and 12 microsatellites. We found that: (a) the Aegypti Group diverged 16 MYA (95% HPD: 7-28 MYA) from its nearest African/Asian ancestor; (b) SWIO populations of Ae. aegypti are basal to continental African populations; (c) after diverging 7 MYA (95% HPD: 4-15 MYA) from its nearest formally described relative (Ae. mascarensis), Ae. aegypti moved to continental Africa less than 85,000 years ago, where it recently (<1,000 years ago) split into two recognized subspecies Ae. aegypti formosus and a human commensal, Ae. aegypti aegypti; (d) the Madagascar samples form a clade more distant from all other Ae. aegypti than the named species Ae. mascarensis, implying that Madagascar may harbour a new cryptic species; and (e) there is evidence of introgression between Ae. mascarensis and Ae. aegypti on Réunion, and between the two subspecies elsewhere in the SWIO, a likely consequence of recent introductions of domestic Ae. aegypti aegypti from Asia.

Recent History of Aedes aegypti: Vector Genomics and Epidemiology Records.

Aedes aegypti bears the common name "the yellow fever mosquito," although, today, it is of more concern as the major vector of dengue, chikungunya, and, most recently, Zika viruses. In the present article, we review recent work on the population genetics of this mosquito in efforts to reconstruct its recent (approximately 600 years) history and relate these findings to epidemiological records of occurrences of diseases transmitted by this species. The two sources of information are remarkably congruent. Ae. aegypti was introduced to the New World 400-550 years ago from its ancestral home in West Africa via European slave trade. Ships from the New World returning to their European ports of origin introduced the species to the Mediterranean region around 1800, where it became established until about 1950. The Suez Canal opened in 1869 and Ae. aegypti was introduced into Asia by the 1870s, then on to Australia (1887) and the South Pacific (1904).

Global genetic diversity of Aedes aegypti.

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.

Robustness in population-structure and demographic-inference results derived from the Aedes aegypti genotyping chip and whole-genome sequencing data.

The mosquito Aedes aegypti is the primary vector of many human arboviruses such as dengue, yellow fever, chikungunya, and Zika, which affect millions of people worldwide. Population genetic studies on this mosquito have been important in understanding its invasion pathways and success as a vector of human disease. The Axiom aegypti1 SNP chip was developed from a sample of geographically diverse A. aegypti populations to facilitate genomic studies on this species. We evaluate the utility of the Axiom aegypti1 SNP chip for population genetics and compare it with a low-depth shotgun sequencing approach using mosquitoes from the native (Africa) and invasive ranges (outside Africa). These analyses indicate that results from the SNP chip are highly reproducible and have a higher sensitivity to capture alternative alleles than a low-coverage whole-genome sequencing approach. Although the SNP chip suffers from ascertainment bias, results from population structure, ancestry, demographic, and phylogenetic analyses using the SNP chip were congruent with those derived from low-coverage whole-genome sequencing, and consistent with previous reports on Africa and outside Africa populations using microsatellites. More importantly, we identified a subset of SNPs that can be reliably used to generate merged databases, opening the door to combined analyses. We conclude that the Axiom aegypti1 SNP chip is a convenient, more accurate, low-cost alternative to low-depth whole-genome sequencing for population genetic studies of A. aegypti that do not rely on full allelic frequency spectra. Whole-genome sequencing and SNP chip data can be easily merged, extending the usefulness of both approaches.

Microsatellite-based analysis reveals Aedes aegypti populations in the Kingdom of Saudi Arabia result from colonization by both the ancestral African and the global domestic forms.

The Aedes aegypti (Linnaeus, 1762) mosquito is the main vector of dengue, chikungunya and Zika and is well established today all over the world. The species comprises two forms: the ancestral form found throughout Africa and a global domestic form that spread to the rest of the tropics and subtropics. In Saudi Arabia, A. aegypti has been known in the southwest since 1956, and previous genetic studies clustered A. aegypti from Saudi Arabia with the global domestic form. The purpose of this study was to assess the genetic structure of A. aegypti in Saudi Arabia and determine their geographic origin. Genetic data for 17 microsatellites were collected for A. aegypti ranging from the southwestern highlands of Saudi Arabia on the border of Yemen to the north-west in Madinah region as well as from Thailand and Uganda populations (as representatives of the ancestral African and global domestic forms, respectively). The low but significant level of genetic structuring in Saudi Arabia was consistent with long-distance dispersal capability possibly through road connectivity and human activities, that is, passive dispersal. There are two main genetic groupings in Saudi Arabia, one of which clusters with the Ugandan population and the other with the Thailand population with many Saudi Arabian individuals having mixed ancestry. The hypothesis of genetic admixture of the ancestral African and global domestic forms in Saudi Arabia was supported by approximate Bayesian computational analyses. The extent of admixture varied across Saudi Arabia. African ancestry was highest in the highland area of the Jazan region followed by the lowland Jazan and Sahil regions. Conversely, the western (Makkah, Jeddah and Madinah) and Najran populations corresponded to the global domesticated form. Given potential differences between the forms in transmission capability, ecology and behaviour, the findings here should be taken into account in vector control efforts in Saudi Arabia.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Evolutionary genetics of the hydroid allodeterminant alr2.

We surveyed genetic variation in alr2, an allodeterminant of the colonial hydroid Hydractinia symbiolongicarpus. We generated cDNA from a sample of 239 Hydractinia colonies collected at Lighthouse Point, Connecticut, and identified 473 alr2 alleles, 198 of which were unique. Rarefaction analysis suggested that the sample was near saturation. Most alleles were rare, with 86% occurring at frequencies of 1% or less. Alleles were highly variable, diverging on average by 18% of the amino acids in a predicted extracellular domain of the molecule. Analysis of 152 full-length alleles confirmed the existence of two structural types, defined by exons 4-8 of the gene. Several residues of the predicted immunoglobulin superfamily-like domains display signatures of positive selection. We also identified 77 unique alr2 pseudogene sequences from 85 colonies. Twenty-seven of these sequences matched expressed alr2 sequences from other colonies. This observation is consistent with pseudogenes contributing to alr2 diversification through sequence donation. A more limited collection of animals was made from a distant, relict population of H. symbiolongicarpus. Sixty percent of the unique sequences identified in this sample were found to match sequences from the Lighthouse Point population. The large number of alr2 alleles, their degree of divergence, the predominance of rare alleles in the population, their persistence over broad spatial and temporal scales, and the signatures of positive selection in multiple residues of the putative recognition domain paint a consistent picture of negative-frequency-dependent selection operating in this system. The genetic diversity observed at alr2 is comparable to that of the most highly polymorphic genetic systems known to date.

Dating the origin and spread of specialization on human hosts in Aedes aegypti mosquitoes.

The globally invasive mosquito subspecies Aedes aegypti aegypti is an effective vector of human arboviruses, in part because it specializes in biting humans and breeding in human habitats. Recent work suggests that specialization first arose as an adaptation to long, hot dry seasons in the West African Sahel, where Ae. aegypti relies on human-stored water for breeding. Here, we use whole-genome cross-coalescent analysis to date the emergence of human-specialist populationsand thus further probe the climate hypothesis. Importantly, we take advantage of the known migration of specialists out of Africa during the Atlantic Slave Trade to calibrate the coalescent clock and thus obtain a more precise estimate of the older evolutionary event than would otherwise be possible. We find that human-specialist mosquitoes diverged rapidly from ecological generalists approximately 5000 years ago, at the end of the African Humid Period-a time when the Sahara dried and water stored by humans became a uniquely stable, aquatic niche in the Sahel. We also use population genomic analyses to date a previously observed influx of human-specialist alleles into major West African cities. The characteristic length of tracts of human-specialist ancestry present on a generalist genetic background in Kumasi and Ouagadougou suggests the change in behavior occurred during rapid urbanization over the last 20-40 years. Taken together, we show that the timing and ecological context of two previously observed shifts towards human biting in Ae. aegypti differ; climate was likely the original driver, but urbanization has become increasingly important in recent decades.

Phylogenomics reveals the history of host use in mosquitoes.

Mosquitoes have profoundly affected human history and continue to threaten human health through the transmission of a diverse array of pathogens. The phylogeny of mosquitoes has remained poorly characterized due to difficulty in taxonomic sampling and limited availability of genomic data beyond the most important vector species. Here, we used phylogenomic analysis of 709 single copy ortholog groups from 256 mosquito species to produce a strongly supported phylogeny that resolves the position of the major disease vector species and the major mosquito lineages. Our analyses support an origin of mosquitoes in the early Triassic (217 MYA [highest posterior density region: 188-250 MYA]), considerably older than previous estimates. Moreover, we utilize an extensive database of host associations for mosquitoes to show that mosquitoes have shifted to feeding upon the blood of mammals numerous times, and that mosquito diversification and host-use patterns within major lineages appear to coincide in earth history both with major continental drift events and with the diversification of vertebrate classes.

Evidence for serial founder events during the colonization of North America by the yellow fever mosquito, Aedes aegypti.

The Aedes aegypti mosquito first invaded the Americas about 500 years ago and today is a widely distributed invasive species and the primary vector for viruses causing dengue, chikungunya, Zika, and yellow fever. Here, we test the hypothesis that the North American colonization by Ae. aegypti occurred via a series of founder events. We present findings on genetic diversity, structure, and demographic history using data from 70 Ae. aegypti populations in North America that were genotyped at 12 microsatellite loci and/or ~20,000 single nucleotide polymorphisms, the largest genetic study of the region to date. We find evidence consistent with colonization driven by serial founder effect (SFE), with Florida as the putative source for a series of westward invasions. This scenario was supported by (1) a decrease in the genetic diversity of Ae. aegypti populations moving west, (2) a correlation between pairwise genetic and geographic distances, and (3) demographic analysis based on allele frequencies. A few Ae. aegypti populations on the west coast do not follow the general trend, likely due to a recent and distinct invasion history. We argue that SFE provides a helpful albeit simplified model for the movement of Ae. aegypti across North America, with outlier populations warranting further investigation.

An alien in Marseille: investigations on a single Aedes aegypti mosquito likely introduced by a merchant ship from tropical Africa to Europe.

Control of invasive species relies partly on permanent surveillance at international points of entry. We report the exceptional trapping of one adult mosquito (Diptera: Culicidae) in the port of Marseille, France, in July 2018, during a routine survey conducted according to International Health Regulations. Morphological and molecular identification classified the specimen as a female Aedes (Stegomyia) aegypti (L.), vector of many arboviruses, absent from Europe and the Mediterranean rim since the 1950s. A world reference panel of approximately 23,000 genome-wide single nucleotide polymorphisms determined that the mosquito originated from Cameroon, west Africa. Cross-reference of this geographic location with boats traveling from Central Africa to Marseille during the trapping period suggests that the mosquito travelled within an identified merchant ship, a vehicles carrier connecting Douala, Cameroon to Marseille, France. This ship left Douala on June 25, 2018 and arrived 20 days later in Marseille on July 15. The mosquito was captured 350 m away from the dock. The interception of a propagule of an invasive species is a rare event that must be considered a priority to prevent its successful establishment.

Title: Un alien à Marseille : enquêtes sur un seul moustique Aedes aegypti vraisemblablement introduit par un navire marchand de l'Afrique tropicale vers l'Europe. Abstract: La lutte contre les espèces invasives repose en partie sur une surveillance permanente aux points d'entrée internationaux. Nous rapportons ici le piégeage exceptionnel d'un moustique adulte (Diptera: Culicidae) dans le port de Marseille, France, en juillet 2018, au cours d'une enquête de routine menée selon les recommandations du Règlement Sanitaire International. L'identification morphologique et moléculaire a désigné ce spécimen comme étant une femelle d'Aedes (Stegomyia) aegypti (L.), vecteur de nombreux arbovirus, absent d'Europe et du pourtour Méditerranéen depuis les années 1950. Une base de référence mondiale du polymorphisme des nucléotides individuels pour ~23 000 génomes complets a permis de déterminer que ce moustique était originaire du Cameroun. Le croisement de cette information de localisation géographique avec celle de la circulation des bateaux entre l'Afrique Centrale et Marseille au cours de la période de piégeage suggère que le moustique a voyagé à l'intérieur d'un navire de commerce identifié, un transporteur de véhicules reliant Douala (Cameroun) à Marseille (France). Ce navire a quitté Douala le 25 juin 2018 pour arriver à Marseille 20 jours plus tard, le 15 juillet 2018. Le moustique a été capturé à 350 mètres du dock. L'interception d'un propagule d'une espèce invasive est un évènement rare qui doit être considéré de façon prioritaire afin d'empêcher la réussite de son installation.

Population Genetic Analysis of Aedes aegypti Mosquitoes From Sudan Revealed Recent Independent Colonization Events by the Two Subspecies.

Increases in arbovirus outbreaks in Sudan are vectored by Aedes aegypti, raising the medical importance of this mosquito. We genotyped 12 microsatellite loci in four populations of Ae. aegypti from Sudan, two from the East and two from the West, and analyzed them together with a previously published database of 31 worldwide populations to infer population structure and investigate the demographic history of this species in Sudan. Our results revealed the presence of two genetically distinct subspecies of Ae. aegypti in Sudan. These are Ae. aegypti aegypti in Eastern Sudan and Ae. aegypti formosus in Western Sudan. Clustering analysis showed that mosquitoes from East Sudan are genetically homogeneous, while we found population substructure in West Sudan. In the global context our results indicate that Eastern Sudan populations are genetically closer to Asian and American populations, while Western Sudan populations are related to East and West African populations. Approximate Bayesian Computation Analysis supports a scenario in which Ae. aegypti entered Sudan in at least two independent occasions nearly 70-80 years ago. This study provides a baseline database that can be used to determine the likely origin of new introductions for this invasive species into Sudan. The presence of the two subspecies in the country should be consider when designing interventions, since they display different behaviors regarding epidemiologically relevant parameters, such as blood feeding preferences and ability to transmit disease.

Origins of high latitude introductions of Aedes aegypti to Nebraska and Utah during 2019.

Aedes aegypti (L.), the yellow fever mosquito, is also an important vector of dengue and Zika viruses, and an invasive species in North America. Aedes aegypti inhabits tropical and sub-tropical areas of the world and in North America is primarily distributed throughout the southern US states and Mexico. The northern range of Ae. aegypti is limited by cold winter months and establishment in these areas has been mostly unsuccessful. However, frequent introductions of Ae. aegypti to temperate, non-endemic areas during the warmer months can lead to seasonal activity and disease outbreaks. Two Ae. aegypti incursions were reported in the late summer of 2019 into York, Nebraska and Moab, Utah. These states had no history of established populations of this mosquito and no evidence of previous seasonal activity. We genotyped a subset of individuals from each location at 12 microsatellite loci and ~ 14,000 single nucleotide polymorphic markers to determine their genetic affinities to other populations worldwide and investigate their potential source of introduction. Our results support a single origin for each of the introductions from different sources. Aedes aegypti from Utah likely derived from Tucson, Arizona, or a nearby location. Nebraska specimen results were not as conclusive, but point to an origin from southcentral or southeastern US. In addition to an effective, efficient, and sustainable control of invasive mosquitoes, such as Ae. aegypti, identifying the potential routes of introduction will be key to prevent future incursions and assess their potential health threat based on the ability of the source population to transmit a particular virus and its insecticide resistance profile, which may complicate vector control.

Population genetics of an invasive mosquito vector, Aedes albopictus in the Northeastern USA.

The Asian tiger mosquito (Aedes albopictus) arrived in the USA in the 1980's and rapidly spread throughout eastern USA within a decade. The predicted northern edge of its overwintering distribution on the East Coast of the USA roughly falls across New York, Connecticut, and Massachusetts, where the species has been recorded as early as 2000. It is unclear whether Ae. albopictus populations have become established and survive the cold winters in these areas or are recolonized every year. We genotyped and analyzed populations of Ae. albopictus from the northeast USA using 15 microsatellite markers and compared them with other populations across the country and to representatives of the major global genetic clades to investigate their connectivity and stability. Founder effects or bottlenecks were rare at the northern range of the Ae. albopictus distribution in the northeastern USA, with populations displaying high levels of genetic diversity and connectivity along the East Coast. There is no evidence of population turnover in Connecticut during the course of three consecutive years, with consistent genetic structure throughout this period. Overall, these results support the presence of established populations of Ae. albopictus in New York, Connecticut, and Massachusetts, successfully overwintering and migrating in large numbers. Given the stability and interconnectedness of these populations, Ae. albopictus has the potential to continue to proliferate and expand its range northward under mean warming conditions of climate change. Efforts to control Ae. albopictus in these areas should thus focus on vector suppression rather than eradication strategies, as local populations have become firmly established and are expected to reemerge every summer.

npr-1 Regulates foraging and dispersal strategies in Caenorhabditis elegans.

Wild isolates of Caenorhabditis elegans differ in their tendency to aggregate on food [1, 2]. Most quantitative variation in this behavior is explained by a polymorphism at a single amino acid in the G protein-coupled receptor NPR-1: gregarious strains carry the 215F allele, and solitary strains carry the 215V allele [2]. Although npr-1 regulates a behavioral syndrome with potential adaptive implications, the evolutionary causes and consequences of this natural polymorphism remain unclear. Here we show that npr-1 regulates two behaviors that can promote coexistence of the two alleles. First, gregarious and solitary worms differ in their responses to food such that they can partition a single, continuous patch of food. Second, gregarious worms disperse more readily from patch to patch than do solitary worms, which can cause partitioning of a fragmented resource. The dispersal propensity of both gregarious and solitary worms increases with density. npr-1-dependent dispersal is independent of aggregation and could be part of a food-searching strategy. The gregarious allele is favored in a fragmented relative to a continuous food environment in competition experiments. We conclude that the npr-1 polymorphism could be maintained by a trade-off between dispersal and competitive ability.

Evolution of kdr haplotypes in worldwide populations of Aedes aegypti: Independent origins of the F1534C kdr mutation.

Aedes aegypti is the primary vector of dengue, chikungunya, Zika, and urban yellow fever. Insecticides are often the most effective tools to rapidly decrease the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has selected for resistant mosquito populations worldwide. Mutations in the voltage gated sodium channel (NaV) are among the principal mechanisms of resistance to pyrethroids and DDT, also known as "knockdown resistance," kdr. Here we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. aegypti from its worldwide distribution. We amplified the IIS6 and IIIS6 NaV segments from pools of Ae. aegypti populations from 15 countries, in South and North America, Africa, Asia, Pacific, and Australia. The amplicons were barcoded and sequenced using NGS Ion Torrent. Output data were filtered and analyzed using the bioinformatic pipeline Seekdeep to determine frequencies of the IIS6 and IIIS6 haplotypes per population. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations have a single or multiple origin. We found 26 and 18 haplotypes, respectively for the IIS6 and IIIS6 segments, among which were the known kdr mutations 989P, 1011M, 1016I and 1016G (IIS6), 1520I, and 1534C (IIIS6). The highest diversity of haplotypes was found in African samples. Kdr mutations 1011M and 1016I were found only in American and African populations, 989P + 1016G and 1520I + 1534C in Asia, while 1534C was present in samples from all continents, except Australia. Based primarily on the intron sequence, IIS6 haplotypes were subdivided into two well-defined clades (A and B). Subsequent phasing of the IIS6 + IIIS6 haplotypes indicates two distinct origins for the 1534C kdr mutation. These results provide evidence of kdr mutations arising de novo at specific locations within the Ae. aegypti geographic distribution. In addition, our results suggest that the 1534C kdr mutation had at least two independent origins. We can thus conclude that insecticide selection pressure with DDT and more recently with pyrethroids is selecting for independent convergent mutations in NaV.

Prior evolution in stochastic versus constant temperatures affects RNA virus evolvability at a thermal extreme.

It is unclear how historical adaptation versus maladaptation in a prior environment affects population evolvability in a novel habitat. Prior work showed that vesicular stomatitis virus (VSV) populations evolved at constant 37°C improved in cellular infection at both 29°C and 37°C; in contrast, those evolved under random changing temperatures between 29°C and 37°C failed to improve. Here, we tested whether prior evolution affected the rate of adaptation at the thermal-niche edge: 40°C. After 40 virus generations in the new environment, we observed that populations historically evolved at random temperatures showed greater adaptability. Deep sequencing revealed that most of the newly evolved mutations were de novo. Also, two novel evolved mutations in the VSV glycoprotein and replicase genes tended to co-occur in the populations previously evolved at constant 37°C, whereas this parallelism was not seen in populations with prior random temperature evolution. These results suggest that prior adaptation under constant versus random temperatures constrained the mutation landscape that could improve fitness in the novel 40°C environment, perhaps owing to differing epistatic effects of new mutations entering genetic architectures that earlier diverged. We concluded that RNA viruses maladapted to their previous environment could "leapfrog" over counterparts of higher fitness, to achieve faster adaptability in a novel environment.

Vector Competence of Aedes albopictus Populations from the Northeastern United States for Chikungunya, Dengue, and Zika Viruses.

The Asian tiger mosquito (Aedes albopictus) is an important vector of a number of arboviruses, including Zika (ZIKV), dengue (DENV), and chikungunya (CHIKV) viruses, and has recently expanded its range in the eastern United States to southern New England and New York. Given the recent establishment and proliferation of Ae. albopictus in this region and the increasing amount of international travel between the United States and endemic countries, there is a need to elucidate the public health risk posed by this mosquito species in the Northeast. Accordingly, we evaluated the competence of four Ae. albopictus populations from Connecticut and New York, for two strains each of ZIKV, DENV serotype 2 (DENV-2), and CHIKV, currently circulating in the Americas, to evaluate the local transmission risk by this vector. We found that local Ae. albopictus populations are susceptible to infection by all three viruses but are most capable of transmitting CHIKV. Variation in competence was observed for ZIKV and CHIKV, driven by the virus strains and mosquito population, whereas competence was more homogeneous for the DENV-2 strains under evaluation. These results suggest that under optimal circumstances, Ae. albopictus could support localized transmission of these viruses and emphasize the importance of maintaining mosquito surveillance and control programs to suppress Ae. albopictus populations and limit further range expansion of this species.

Novel genome sequences of cell-fusing agent virus allow comparison of virus phylogeny with the genetic structure of Aedes aegypti populations.

Flaviviruses encompass not only medically relevant arthropod-borne viruses (arboviruses) but also insect-specific flaviviruses (ISFs) that are presumably maintained primarily through vertical transmission in the insect host. Interestingly, ISFs are commonly found infecting important arbovirus vectors such as the mosquito Aedes aegypti. Cell-fusing agent virus (CFAV) was the first described ISF of mosquitoes more than four decades ago. Despite evidence for widespread CFAV infections in A.aegypti populations and for CFAV potential to interfere with arbovirus transmission, little is known about CFAV evolutionary history. Here, we generated six novel CFAV genome sequences by sequencing three new virus isolates and subjecting three mosquito samples to untargeted viral metagenomics. We used these new genome sequences together with published ones to perform a global phylogenetic analysis of CFAV genetic diversity. Although there was some degree of geographical clustering among CFAV sequences, there were also notable discrepancies between geography and phylogeny. In particular, CFAV sequences from Cambodia and Thailand diverged significantly, despite confirmation that A.aegypti populations from both locations are genetically close. The apparent phylogenetic discrepancy between CFAV and its A.aegypti host in Southeast Asia indicates that other factors than host population structure shape CFAV genetic diversity.