Mosquito brains encode unique features of human odour to drive host seeking.

A globally invasive form of the mosquito Aedes aegypti specializes in biting humans, making it an efficient disease vector1. Host-seeking female mosquitoes strongly prefer human odour over the odour of animals2,3, but exactly how they distinguish between the two is not known. Vertebrate odours are complex blends of volatile chemicals with many shared components4-7, making discrimination an interesting sensory coding challenge. Here we show that human and animal odours evoke activity in distinct combinations of olfactory glomeruli within the Ae. aegypti antennal lobe. One glomerulus in particular is strongly activated by human odour but responds weakly, or not at all, to animal odour. This human-sensitive glomerulus is selectively tuned to the long-chain aldehydes decanal and undecanal, which we show are consistently enriched in human odour and which probably originate from unique human skin lipids. Using synthetic blends, we further demonstrate that signalling in the human-sensitive glomerulus significantly enhances long-range host-seeking behaviour in a wind tunnel, recapitulating preference for human over animal odours. Our research suggests that animal brains may distil complex odour stimuli of innate biological relevance into simple neural codes and reveals targets for the design of next-generation mosquito-control strategies.

Enhanced mosquito vectorial capacity underlies the Cape Verde Zika epidemic.

The explosive emergence of Zika virus (ZIKV) across the Pacific and Americas since 2007 was associated with hundreds of thousands of human cases and severe outcomes, including congenital microcephaly caused by ZIKV infection during pregnancy. Although ZIKV was first isolated in Uganda, Africa has so far been exempt from large-scale ZIKV epidemics, despite widespread susceptibility among African human populations. A possible explanation for this pattern is natural variation among populations of the primary vector of ZIKV, the mosquito Aedes aegypti. Globally invasive populations of Ae. aegypti outside of Africa are considered effective ZIKV vectors because they are human specialists with high intrinsic ZIKV susceptibility, whereas African populations of Ae. aegypti across the species' native range are predominantly generalists with low intrinsic ZIKV susceptibility, making them less likely to spread viruses in the human population. We test this idea by studying a notable exception to the patterns observed across most of Africa: Cape Verde experienced a large ZIKV outbreak in 2015 to 2016. We find that local Ae. aegypti in Cape Verde have substantial human-specialist ancestry, show a robust behavioral preference for human hosts, and exhibit increased susceptibility to ZIKV infection, consistent with a key role for variation among mosquito populations in ZIKV epidemiology. These findings suggest that similar human-specialist populations of Ae. aegypti in the nearby Sahel region of West Africa, which may be expanding in response to rapid urbanization, could serve as effective vectors for ZIKV in the future.

The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes.

BACKGROUND: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. METHODS: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. RESULTS: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. CONCLUSION: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.

Evolution of a Mosquito's Hatching Behavior to Match Its Human-Provided Habitat.

AbstractA subspecies of the yellow fever mosquito, Aedes aegypti, has recently evolved to specialize in biting and living alongside humans. It prefers human odor over the odor of nonhuman animals and breeds in human-provided artificial containers rather than the forest tree holes of its ancestors. Here, we report one way this human specialist has adapted to the distinct ecology of human environments. While eggs of the ancestral subspecies rarely hatch in pure water, those of the derived human specialist do so readily. We trace this novel behavior to a shift in how eggs respond to dissolved oxygen, low levels of which may signal food abundance. Moreover, we show that while tree holes are consistently low in dissolved oxygen, artificial containers often have much higher levels. There is thus a concordance between the hatching behavior of each subspecies and the aquatic habitat it uses in the wild. We find this behavioral variation is heritable, with both maternal and zygotic effects. The zygotic effect depends on dissolved oxygen concentration (i.e., a genotype-environment interaction, or G×E), pointing to potential changes in oxygen-sensitive circuits. Together, our results suggest that a shift in hatching response contributed to the pernicious success of this human-specialist mosquito and illustrate how animals may rapidly adapt to human-driven changes in the environment.

Correction to: Evolution of olfactory circuits in insects.

Authors would like to correct the typo found in the fourth sentence of the introduction from "organisional" to "organismal". The original article has been corrected.

Evolution of olfactory circuits in insects.

Recent years have seen an explosion of interest in the evolution of neural circuits. Comparison of animals from different families, orders, and phyla reveals fascinating variation in brain morphology, circuit structure, and neural cell types. However, it can be difficult to connect the complex changes that occur across long evolutionary distances to behavior. Luckily, these changes accumulate through processes that should also be observable in recent time, making more tractable comparisons of closely related species relevant and complementary. Here, we review several decades of research on the evolution of insect olfactory circuits across short evolutionary time scales. We describe two well-studied systems, Drosophila sechellia flies and Heliothis moths, in detailed case studies. We then move through key types of circuit evolution, cataloging examples from other insects and looking for general patterns. The literature is dominated by changes in sensory neuron number and tuning at the periphery-often enhancing neural response to odorants with new ecological or social relevance. However, changes in the way olfactory information is processed by central circuits is clearly important in a few cases, and we suspect the development of genetic tools in non-model species will reveal a broad role for central circuit evolution. Moving forward, such tools should also be used to rigorously test causal links between brain evolution and behavior.

Evolution of mosquito preference for humans linked to an odorant receptor.

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A 'domestic' form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, 'forest' form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET.

Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever. Specific host odours probably drive this strong preference because other attractive cues, including body heat and exhaled carbon dioxide (CO2), are common to all warm-blooded hosts. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs), membrane proteins that form heteromeric odour-gated ion channels comprising a variable ligand-selective subunit and an obligate co-receptor called Orco (ref. 6). Here we use zinc-finger nucleases to generate targeted mutations in the orco gene of A. aegypti to examine the contribution of Orco and the odorant receptor pathway to mosquito host selection and sensitivity to the insect repellent DEET (N,N-diethyl-meta-toluamide). orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the odorant receptor pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.

The neurotranscriptome of the Aedes aegypti mosquito.

BACKGROUND: A complete genome sequence and the advent of genome editing open up non-traditional model organisms to mechanistic genetic studies. The mosquito Aedes aegypti is an important vector of infectious diseases such as dengue, chikungunya, and yellow fever and has a large and complex genome, which has slowed annotation efforts. We used comprehensive transcriptomic analysis of adult gene expression to improve the genome annotation and to provide a detailed tissue-specific catalogue of neural gene expression at different adult behavioral states. RESULTS: We carried out deep RNA sequencing across all major peripheral male and female sensory tissues, the brain and (female) ovary. Furthermore, we examined gene expression across three important phases of the female reproductive cycle, a remarkable example of behavioral switching in which a female mosquito alternates between obtaining blood-meals from humans and laying eggs. Using genome-guided alignments and de novo transcriptome assembly, our re-annotation includes 572 new putative protein-coding genes and updates to 13.5 and 50.3 % of existing transcripts within coding sequences and untranslated regions, respectively. Using this updated annotation, we detail gene expression in each tissue, identifying large numbers of transcripts regulated by blood-feeding and sexually dimorphic transcripts that may provide clues to the biology of male- and female-specific behaviors, such as mating and blood-feeding, which are areas of intensive study for those interested in vector control. CONCLUSIONS: This neurotranscriptome forms a strong foundation for the study of genes in the mosquito nervous system and investigation of sensory-driven behaviors and their regulation. Furthermore, understanding the molecular genetic basis of mosquito chemosensory behavior has important implications for vector control.

Thermal tolerance of mosquito eggs is associated with urban adaptation and human interactions.

Climate change is expected to profoundly affect mosquito distributions and their ability to serve as vectors for disease, specifically with the anticipated increase in heat waves. The rising temperature and frequent heat waves can accelerate mosquito life cycles, facilitating higher disease transmission. Conversely, higher temperatures could increase mosquito mortality as a negative consequence. Warmer temperatures are associated with increased human density, suggesting a need for anthropophilic mosquitoes to adapt to be more hardy to heat stress. Mosquito eggs provide an opportunity to study the biological impact of climate warming as this stage is stationary and must tolerate temperatures at the site of female oviposition. As such, egg thermotolerance is critical for survival in a specific habitat. In nature, Aedes mosquitoes exhibit different behavioral phenotypes, where specific populations prefer depositing eggs in tree holes and prefer feeding non-human vertebrates. In contrast, others, particularly human-biting specialists, favor laying eggs in artificial containers near human dwellings. This study examined the thermotolerance of eggs, along with adult stages, for Aedes aegypti and Ae. albopictus lineages associated with known ancestry and shifts in their relationship with humans. Mosquitoes collected from areas with higher human population density, displaying increased human preference, and having a human-associated ancestry profile have increased egg viability following high-temperature stress. Unlike eggs, thermal tolerance among adults showed no significant correlation based on the area of collection or human-associated ancestry. This study highlights that the egg stage is likely critical to mosquito survival when associated with humans and needs to be accounted when predicting future mosquito distribution.

Extensive variation and strain-specificity in dengue virus susceptibility among African Aedes aegypti populations.

African populations of the mosquito Aedes aegypti are usually considered less susceptible to infection by human-pathogenic flaviviruses than globally invasive populations found outside Africa. Although this contrast has been well documented for Zika virus (ZIKV), it is unclear to what extent it is true for dengue virus (DENV), the most prevalent flavivirus of humans. Addressing this question is complicated by substantial genetic diversity among DENV strains, most notably in the form of four genetic types (DENV1 to DENV4), that can lead to genetically specific interactions with mosquito populations. Here, we carried out a survey of DENV susceptibility using a panel of seven field-derived Ae. aegypti colonies from across the African range of the species and a colony from Guadeloupe, French West Indies as non-African reference. We found considerable variation in the ability of African Ae. aegypti populations to acquire and replicate a panel of six DENV strains spanning the four DENV types. Although African Ae. aegypti populations were generally less susceptible than the reference non-African population from Guadeloupe, in several instances some African populations were equally or more susceptible than the Guadeloupe population. Moreover, the relative level of susceptibility between African mosquito populations depended on the DENV strain, indicating genetically specific interactions. We conclude that unlike ZIKV susceptibility, there is no clear-cut dichotomy in DENV susceptibility between African and non-African Ae. aegypti. DENV susceptibility of African Ae. aegypti populations is highly heterogeneous and largely governed by the specific pairing of mosquito population and DENV strain.

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.

Host-associated genomic differentiation in congeneric butterflies: now you see it, now you do not.

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host-associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host-adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host-adapted traits, resulting in incipient reproductive isolation among host-associated ecotypes. We show here that Euphydryas aurinia had much weaker host-associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host-adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation.

Field studies reveal strong postmating isolation between ecologically divergent butterfly populations.

Gene flow between populations that are adapting to distinct environments may be restricted if hybrids inherit maladaptive, intermediate phenotypes. This phenomenon, called extrinsic postzygotic isolation (EPI), is thought to play a critical role in the early stages of speciation. However, despite its intuitive appeal, we know surprisingly little about the strength and prevalence of EPI in nature, and even less about the specific phenotypes that tend to cause problems for hybrids. In this study, we searched for EPI among allopatric populations of the butterfly Euphydryas editha that have specialized on alternative host plants. These populations recall a situation thought typical of the very early stages of speciation. They lack consistent host-associated genetic differentiation at random nuclear loci and show no signs of reproductive incompatibility in the laboratory. However, they do differ consistently in diverse host-related traits. For each of these traits, we first asked whether hybrids between populations that use different hosts (different-host hybrids) were intermediate to parental populations and to hybrids between populations that use the same host (same-host hybrids). We then conducted field experiments to estimate the effects of intermediacy on fitness in nature. Our results revealed strong EPI under field conditions. Different-host hybrids exhibited an array of intermediate traits that were significantly maladaptive, including four behaviors. Intermediate foraging height slowed the growth of larvae, while intermediate oviposition preference, oviposition site height, and clutch size severely reduced the growth and survival of the offspring of adult females. We used our empirical data to construct a fitness surface on which different-host hybrids can be seen to fall in an adaptive valley between two peaks occupied by same-host hybrids. These findings demonstrate how ecological selection against hybrids can create a strong barrier to gene flow at the early stages of adaptive divergence.

Exploring natural odour landscapes: A case study with implications for human-biting insects.

The natural world is full of odours-blends of volatile chemicals emitted by potential sources of food, social partners, predators, and pathogens. Animals rely heavily on these signals for survival and reproduction. Yet we remain remarkably ignorant of the composition of the chemical world. How many compounds do natural odours typically contain? How often are those compounds shared across stimuli? What are the best statistical strategies for discrimination? Answering these questions will deliver crucial insight into how brains can most efficiently encode olfactory information. Here, we undertake the first large-scale survey of vertebrate body odours, a set of stimuli relevant to blood-feeding arthropods. We quantitatively characterize the odour of 64 vertebrate species (mostly mammals), representing 29 families and 13 orders. We confirm that these stimuli are complex blends of relatively common, shared compounds and show that they are much less likely to contain unique components than are floral odours-a finding with implications for olfactory coding in blood feeders and floral visitors. We also find that vertebrate body odours carry little phylogenetic information, yet show consistency within a species. Human odour is especially unique, even compared to the odour of other great apes. Finally, we use our newfound understanding of odour-space statistics to make specific predictions about olfactory coding, which align with known features of mosquito olfactory systems. Our work provides one of the first quantitative descriptions of a natural odour space and demonstrates how understanding the statistics of sensory environments can provide novel insight into sensory coding and evolution.

Two-Photon Calcium Imaging in the Brain of Aedes aegypti Mosquitoes.

Understanding the neural basis of mosquito behavior is critical for designing effective vector control strategies and can potentially shed new light on basic nervous system function. Because mosquitoes are a non-model species, however, functional studies of mosquito nervous systems have long been restricted to electrophysiological recording from peripheral sensory organs such as the antenna. This is now changing with the advent of CRISPR-Cas9 gene editing and the development of other powerful new genetic tools. Transgenic mosquitoes that carry genetically encoded calcium sensors, for example, open the door to optical recording of neural activity with two-photon calcium imaging. Compared with electrophysiology, calcium imaging permits continuous monitoring of neural activity from large populations of neurons, even deep in the brain. When combined with selective neural drivers, it also allows targeted recording from specific neuronal types. Here, we describe a calcium imaging protocol we use in our laboratory to study neural activity in the brain of Aedes aegypti mosquitoes.

GCaMP Imaging in Mosquitoes: Central Nervous System.

Mosquitoes spread dengue, Zika, malaria, and other pathogens to hundreds of millions of people every year. A better understanding of mosquito behavior and its underlying neural mechanisms may lead to new control strategies, but such an understanding requires the development of tools and approaches for exploring the nervous system of key vector species. For example, we can now image neural activity in mosquito brains using genetically encoded calcium sensors like GCaMP. Compared with other types of neural recording, GCaMP imaging has the advantage of allowing one to record from many neurons simultaneously and/or to record from specific neuronal types. Successful implementation requires careful consideration of many factors, including the choice of microscope and how to make the brains of experimental animals visible and stable while minimizing damage. Here, we elaborate on these points and provide a concise introduction to GCaMP imaging in the mosquito central nervous system.

Quantifying Aedes aegypti Host Odor Preference Using a Two-Port Olfactometer.

Blood-feeding mosquitoes are a leading threat to global public health-vectoring dangerous infections including Zika, dengue, and malaria. Mosquitoes identify and target hosts for blood meals by using visual, thermal, and chemical cues. Here we describe an assay for measuring odor-based host-preference behavior-that is, the preferential approach toward one host over another based on differences in the volatile compounds they emit. The assay can be adapted for use with diverse odor sources, from live animals and their breath to odor-scented sleeves with controlled amounts of CO2 Mosquitoes in this assay fly upwind to within 30 cm of the odor source and then enter a small trap. We therefore believe this assay best replicates medium- to short-range host-seeking, when females approach and are preparing to land on a host animal. We also find that relative response in a two-choice test shows less trial-to-trial variation than the absolute number of responsive mosquitoes, which appears more sensitive to exogenous factors such as rearing conditions. This assay has been used to better understand mosquito host-seeking decisions, which can provide fundamental insight into the brain and behavior as well as information useful for the design of novel vector control strategies.

An Assay for Quantifying Aedes aegypti Host Odor Preference Using a Two-Port Olfactometer.

Female mosquitoes use odor cues to locate hosts for blood meals and are often more likely to approach the odor of certain species or individuals over others. Here, we describe an assay for measuring such odor-based host preference. This assay uses a two-port olfactometer and can be adapted to study a wide variety of odor sources including live hosts, host-scented nylon sleeves or host hair samples, and single odorants or odorant blends.