Next-generation neuropeptide Y receptor small-molecule agonists inhibit mosquito-biting behavior.

BACKGROUND: Female Aedes aegypti mosquitoes can spread disease-causing pathogens when they bite humans to obtain blood nutrients required for egg production. Following a complete blood meal, host-seeking is suppressed until eggs are laid. Neuropeptide Y-like receptor 7 (NPYLR7) plays a role in endogenous host-seeking suppression and previous work identified small-molecule NPYLR7 agonists that inhibit host-seeking and blood-feeding when fed to mosquitoes at high micromolar doses. METHODS: Using structure-activity relationship analysis and structure-guided design we synthesized 128 compounds with similarity to known NPYLR7 agonists. RESULTS: Although in vitro potency (EC50) was not strictly predictive of in vivo effect, we identified three compounds that reduced blood-feeding from a live host when fed to mosquitoes at a dose of 1 µM-a 100-fold improvement over the original reference compound. CONCLUSIONS: Exogenous activation of NPYLR7 represents an innovative vector control strategy to block mosquito biting behavior and prevent mosquito-human host interactions that lead to pathogen transmission.

Next Generation Neuropeptide Y Receptor Small Molecule Agonists Inhibit Mosquito Biting Behavior.

Female Aedes aegypti mosquitoes can spread disease-causing pathogens when they bite humans to obtain blood nutrients required for egg production. Following a complete blood meal, host-seeking is suppressed until eggs are laid. Neuropeptide Y-like Receptor 7 (NPYLR7) plays a role in endogenous host-seeking suppression and previous work identified small molecule NPYLR7 agonists that suppress host-seeking and blood feeding when fed to mosquitoes at high micromolar doses. Using structure activity relationship analysis and structure-guided design we synthesized 128 compounds with similarity to known NPYLR7 agonists. Although in vitro potency (EC50) was not strictly predictive of in vivo effect, we identified 3 compounds that suppressed blood feeding from a live host when fed to mosquitoes at a 1 µM dose, a 100-fold improvement over the original reference compound. Exogenous activation of NPYLR7 represents an innovative vector control strategy to block mosquito biting behavior and prevent mosquito/human host interactions that lead to pathogen transmission.

Two novel, tightly linked, and rapidly evolving genes underlie Aedes aegypti mosquito reproductive resilience during drought.

Female Aedes aegypti mosquitoes impose a severe global public health burden as vectors of multiple viral pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges like intense droughts and intermittent precipitation, which create unpredictable, suboptimal conditions for egg-laying. Here, we show that under drought-like conditions simulated in the laboratory, females retain mature eggs in their ovaries for extended periods, while maintaining the viability of these eggs until they can be laid in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes named tweedledee and tweedledum, each encoding a small, secreted protein that both show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. CRISPR-Cas9 deletion of both tweedledee and tweedledum demonstrates that they are specifically required for extended retention of viable eggs. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with 'insurance' to flexibly extend their reproductive schedule without losing reproductive capacity, thus allowing this species to exploit unpredictable habitats in a changing world.

Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels.

Some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. We tested mosquito attraction to human skin odor and identified people who are exceptionally attractive or unattractive to mosquitoes. These differences were stable over several years. Chemical analysis revealed that highly attractive people produce significantly more carboxylic acids in their skin emanations. Mutant mosquitoes lacking the chemosensory co-receptors Ir8a, Ir25a, or Ir76b were severely impaired in attraction to human scent, but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in "mosquito-magnet" human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others provides insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.

Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

A persistent behavioral state enables sustained predation of humans by mosquitoes.

Predatory animals pursue prey in a noisy sensory landscape, deciding when to continue or abandon their chase. The mosquito Aedes aegypti is a micropredator that first detects humans at a distance through sensory cues such as carbon dioxide. As a mosquito nears its target, it senses more proximal cues such as body heat that guide it to a meal of blood. How long the search for blood continues after initial detection of a human is not known. Here, we show that a 5 s optogenetic pulse of fictive carbon dioxide induced a persistent behavioral state in female mosquitoes that lasted for more than 10 min. This state is highly specific to females searching for a blood meal and was not induced in recently blood-fed females or in males, who do not feed on blood. In males that lack the gene fruitless, which controls persistent social behaviors in other insects, fictive carbon dioxide induced a long-lasting behavior response resembling the predatory state of females. Finally, we show that the persistent state triggered by detection of fictive carbon dioxide enabled females to engorge on a blood meal mimic offered up to 14 min after the initial 5 s stimulus. Our results demonstrate that a persistent internal state allows female mosquitoes to integrate multiple human sensory cues over long timescales, an ability that is key to their success as an apex micropredator of humans.

Fruitless mutant male mosquitoes gain attraction to human odor.

The Aedesaegypti mosquito shows extreme sexual dimorphism in feeding. Only females are attracted to and obtain a blood-meal from humans, which they use to stimulate egg production. The fruitless gene is sex-specifically spliced and encodes a BTB zinc-finger transcription factor proposed to be a master regulator of male courtship and mating behavior across insects. We generated fruitless mutant mosquitoes and showed that males failed to mate, confirming the ancestral function of this gene in male sexual behavior. Remarkably, fruitless males also gain strong attraction to a live human host, a behavior that wild-type males never display, suggesting that male mosquitoes possess the central or peripheral neural circuits required to host-seek and that removing fruitless reveals this latent behavior in males. Our results highlight an unexpected repurposing of a master regulator of male-specific sexual behavior to control one module of female-specific blood-feeding behavior in a deadly vector of infectious diseases.

Sexual dimorphism is a phenomenon among animals, insects and plants where the two sexes of a species show differences in body size, physical features or colors. The bushy mane of a male lion, for example, is nowhere to be seen on a female lioness, and only male peacocks have extravagant tails. Most examples of sexual dimorphism, such as elaborate visual displays or courtship behaviors, are linked to mating. However, there are a few species where behavioral differences between the sexes are not connected to mating. Mosquitoes are an example: while female mosquitoes feed on humans, and are attracted to a person's body heat and odor, male mosquitoes have little interest in biting humans for their blood. Therefore, female mosquitoes are the ones responsible for transmitting the viruses that cause certain blood-borne diseases such as dengue fever or Zika. Determining which genes are linked to feeding behaviors in mosquitoes could allow researchers to genetically engineer females so they no longer bite people, thus stopping the spread of these diseases. Unfortunately, the genes that control mosquito feeding behaviors have not been well studied. In other insects, some of the genes that control mating behaviors that depend on sex have been identified. For example, a gene called fruitless controls courtship behaviors in male flies and silkworms, and is thought to be the 'master regulator' of male sexual behavior across insects. Yet it remains to be seen whether the fruitless gene has any effect in mosquitoes, where sex differences relate to feeding habits. To investigate this, Basrur et al. removed the fruitless gene from Aedes aegypti mosquitoes. The genetically altered male mosquitoes became unable to mate successfully, but ­ similar to unmodified males ­ still preferred sugar water over blood when feeding. Unlike unmodified males, however, the male mosquitoes lacking fruitless were attracted to the body odor of a person's arm (like females). These results reveal that fruitless, a gene that controls sex-specific mating behaviors in other insects, controls a sex-specific feeding behavior in mosquitoes. The fruitless gene, Basrur et al. speculate, likely gained this role controlling mosquito feeding behavior in the course of evolution. More research is required to fully understand the effects of the fruitless gene in male and female mosquitoes.

Sensory Discrimination of Blood and Floral Nectar by Aedes aegypti Mosquitoes.

Blood-feeding mosquitoes survive by feeding on nectar for metabolic energy but require a blood meal to develop eggs. Aedes aegypti females must accurately discriminate blood and nectar because each meal promotes mutually exclusive feeding programs with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. We investigated the syringe-like blood-feeding appendage, the stylet, and discovered that sexually dimorphic stylet neurons taste blood. Using pan-neuronal calcium imaging, we found that blood is detected by four functionally distinct stylet neuron classes, each tuned to specific blood components associated with diverse taste qualities. Stylet neurons are insensitive to nectar-specific sugars and respond to glucose only in the presence of additional blood components. The distinction between blood and nectar is therefore encoded in specialized neurons at the very first level of sensory detection in mosquitoes. This innate ability to recognize blood is the basis of vector-borne disease transmission to millions of people worldwide.

How to turn an organism into a model organism in 10 'easy' steps.

Many of the major biological discoveries of the 20th century were made using just six species: Escherichia coli bacteria, Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast, Caenorhabditis elegans nematodes, Drosophila melanogaster flies and Mus musculus mice. Our molecular understanding of the cell division cycle, embryonic development, biological clocks and metabolism were all obtained through genetic analysis using these species. Yet the 'big 6' did not start out as genetic model organisms (hereafter 'model organisms'), so how did they mature into such powerful systems? First, these model organisms are abundant human commensals: they are the bacteria in our gut, the yeast in our beer and bread, the nematodes in our compost pile, the flies in our kitchen and the mice in our walls. Because of this, they are cheaply, easily and rapidly bred in the laboratory and in addition were amenable to genetic analysis. How and why should we add additional species to this roster? We argue that specialist species will reveal new secrets in important areas of biology and that with modern technological innovations like next-generation sequencing and CRISPR-Cas9 genome editing, the time is ripe to move beyond the big 6. In this review, we chart a 10-step path to this goal, using our own experience with the Aedes aegypti mosquito, which we built into a model organism for neurobiology in one decade. Insights into the biology of this deadly disease vector require that we work with the mosquito itself rather than modeling its biology in another species.

General Visual and Contingent Thermal Cues Interact to Elicit Attraction in Female Aedes aegypti Mosquitoes.

Female Aedes aegypti mosquitoes use multiple sensory modalities to hunt human hosts and obtain a blood meal for egg production. Attractive cues include carbon dioxide (CO2), a major component of exhaled breath [1, 2]; heat elevated above ambient temperature, signifying warm-blooded skin [3, 4]; and dark visual contrast [5, 6], proposed to bridge long-range olfactory and short-range thermal cues [7]. Any of these sensory cues in isolation is an incomplete signal of a human host, and so a mosquito must integrate multimodal sensory information before committing to approaching and biting a person [8]. Here, we study the interaction of visual cues, heat, and CO2 to investigate the contributions of human-associated stimuli to host-seeking decisions. We show that tethered flying mosquitoes strongly orient toward dark visual contrast, regardless of CO2 stimulation and internal host-seeking status. This suggests that attraction to visual contrast is general and not contingent on other host cues. In free-flight experiments with CO2, adding a dark contrasting visual cue to a warmed surface enhanced attraction. Moderate warmth became more attractive to mosquitoes, and mosquitoes aggregated on the cue at all non-noxious temperatures. Gr3 mutants, unable to detect CO2, were lured to the visual cue at ambient temperatures but fled and did not return when the surface was warmed to host-like temperatures. This suggests that attraction to thermal cues is contingent on the presence of the additional sensory cue CO2. Our results illustrate that mosquitoes integrate general attractive visual stimuli with context-dependent thermal stimuli to seek promising sites for blood feeding.

The ion channel ppk301 controls freshwater egg-laying in the mosquito Aedes aegypti.

Female Aedes aegypti mosquitoes are deadly vectors of arboviral pathogens and breed in containers of freshwater associated with human habitation. Because high salinity is lethal to offspring, correctly evaluating water purity is a crucial parenting decision. We found that the DEG/ENaC channel ppk301 and sensory neurons expressing ppk301 control egg-laying initiation and choice in Ae. aegypti. Using calcium imaging, we found that ppk301-expressing cells show ppk301-dependent responses to water but, unexpectedly, also respond to salt in a ppk301-independent fashion. This suggests that ppk301 is instructive for egg-laying at low-salt concentrations, but that a ppk301-independent pathway is responsible for inhibiting egg-laying at high-salt concentrations. Water is a key resource for insect survival and understanding how mosquitoes interact with water to control different behaviors is an opportunity to study the evolution of chemosensory systems.

Aedes aegypti Mosquitoes Use Their Legs to Sense DEET on Contact.

DEET (N, N-diethyl-meta-toluamide) is the most effective and widely used insect repellent, but its mechanism of action is both complex and controversial [1]. DEET acts on insect smell [2-6] and taste [7-11], and its olfactory mode of action requires the odorant co-receptor orco [2, 3, 6]. We previously observed that orco mutant female Aedes aegypti mosquitoes are strongly attracted to humans even in the presence of DEET, but they are rapidly repelled after contacting DEET-treated skin [6]. DEET inhibits food ingestion by Drosophila melanogaster flies, and this repellency is mediated by bitter taste neurons in the proboscis [9]. Similar neurons were identified in the mosquito proboscis, leading to the hypothesis that DEET repels on contact by activating an aversive bitter taste pathway [10]. To understand the basis of DEET contact chemorepellency, we carried out behavioral experiments and discovered that DEET acts by three distinct mechanisms: smell, ingestion, and contact. Like bitter tastants, DEET is a feeding deterrent when ingested, but its bitterness per se does not fully explain DEET contact chemorepellency. Mosquitoes blood fed on human arms treated with high concentrations of bitters, but rapidly avoided DEET-treated skin and did not blood feed. Insects detect tastants both through their proboscis and legs. We show that DEET contact chemorepellency is mediated exclusively by the tarsal segments of the legs and not the proboscis. This work establishes mosquito legs as the behaviorally relevant contact sensors of DEET. These results will inform the search for molecular mechanisms mediating DEET contact chemorepellency and novel contact-based insect repellents.

Small-Molecule Agonists of Ae. aegypti Neuropeptide Y Receptor Block Mosquito Biting.

Female Aedes aegypti mosquitoes bite humans to obtain blood to develop their eggs. Remarkably, their strong attraction to humans is suppressed for days after the blood meal by an unknown mechanism. We investigated a role for neuropeptide Y (NPY)-related signaling in long-term behavioral suppression and discovered that drugs targeting human NPY receptors modulate mosquito host-seeking. In a screen of all 49 predicted Ae. aegypti peptide receptors, we identified NPY-like receptor 7 (NPYLR7) as the sole target of these drugs. To obtain small-molecule agonists selective for NPYLR7, we performed a high-throughput cell-based assay of 265,211 compounds and isolated six highly selective NPYLR7 agonists that inhibit mosquito attraction to humans. NPYLR7 CRISPR-Cas9 null mutants are defective in behavioral suppression and resistant to these drugs. Finally, we show that these drugs can inhibit biting and blood-feeding on a live host, suggesting a novel approach to control infectious disease transmission by controlling mosquito behavior. VIDEO ABSTRACT.

A natural variant and engineered mutation in a GPCR promote DEET resistance in C. elegans.

DEET (N,N-diethyl-meta-toluamide) is a synthetic chemical identified by the US Department of Agriculture in 1946 in a screen for repellents to protect soldiers from mosquito-borne diseases1,2. Since its discovery, DEET has become the world's most widely used arthropod repellent and is effective against invertebrates separated by millions of years of evolution-including biting flies3, honeybees4, ticks5, and land leeches3. In insects, DEET acts on the olfactory system5-12 and requires the olfactory receptor co-receptor Orco7,9-12, but exactly how it works remains controversial13. Here we show that the nematode Caenorhabditis elegans is sensitive to DEET and use this genetically tractable animal to study the mechanism of action of this chemical. We found that DEET is not a volatile repellent, but instead interferes selectively with chemotaxis to a variety of attractant and repellent molecules. In a forward genetic screen for DEET-resistant worms, we identified a gene that encodes a single G protein-coupled receptor, str-217, which is expressed in a single pair of chemosensory neurons that are responsive to DEET, called ADL neurons. Mis-expression of str-217 in another chemosensory neuron conferred responses to DEET. Engineered str-217 mutants, and a wild isolate of C. elegans that carries a str-217 deletion, are resistant to DEET. We found that DEET can interfere with behaviour by inducing an increase in average pause length during locomotion, and show that this increase in pausing requires both str-217 and ADL neurons. Finally, we demonstrated that ADL neurons are activated by DEET and that optogenetic activation of ADL neurons increased average pause length. This is consistent with the 'confusant' hypothesis, which proposes that DEET is not a simple repellent but that it instead modulates multiple olfactory pathways to scramble behavioural responses10,11. Our results suggest a consistent motif in the effectiveness of DEET across widely divergent taxa: an effect on multiple chemosensory neurons that disrupts the pairing between odorant stimulus and behavioural response.

A Peptide Signaling System that Rapidly Enforces Paternity in the Aedes aegypti Mosquito.

Female Aedes aegypti mosquitoes typically mate only once with one male in their lifetime, a behavior known as "monandry" [1]. This single mating event provisions the female with sufficient sperm to fertilize the >500 eggs she will produce during her ∼4- to 6-week lifespan in the laboratory [2]. Successful mating induces lifetime refractoriness to subsequent insemination by other males, enforcing the paternity of the first male [3-5]. Ae. aegypti mate in flight near human hosts [6], and females become refractory to remating within seconds [1, 3, 4], suggesting the existence of a rapid mechanism to prevent female remating. In this study, we implicate HP-I, an Aedes- and male-specific peptide transferred to females [7], and its cognate receptor in the female, NPYLR1 [8], in rapid enforcement of paternity. HP-I mutant males were ineffective in enforcing paternity when a second male was given access to the female within 1 hr. NPYLR1 mutant females produced mixed paternity offspring at high frequency, indicating acceptance of multiple mates. Synthetic HP-I injected into wild-type, but not NPYLR1 mutant, virgins reduced successful matings. Asian tiger mosquito (Ae. albopictus) HP-I peptides potently activated Ae. aegypti NPYLR1. Invasive Ae. albopictus males are known to copulate with and effectively sterilize Ae. aegypti females by causing them to reject future mates [9]. Cross-species transfer of sperm and active seminal fluid proteins including HP-I may contribute to this phenomenon. This signaling system promotes rapid paternity enforcement within Ae. aegypti but may promote local extinction in areas where they compete with Ae. albopictus.

SMELL-S and SMELL-R: Olfactory tests not influenced by odor-specific insensitivity or prior olfactory experience.

Smell dysfunction is a common and underdiagnosed medical condition that can have serious consequences. It is also an early biomarker of neurodegenerative diseases, including Alzheimer's disease, where olfactory deficits precede detectable memory loss. Clinical tests that evaluate the sense of smell face two major challenges. First, human sensitivity to individual odorants varies significantly, so test results may be unreliable in people with low sensitivity to a test odorant but an otherwise normal sense of smell. Second, prior familiarity with odor stimuli can bias smell test performance. We have developed nonsemantic tests for olfactory sensitivity (SMELL-S) and olfactory resolution (SMELL-R) that use mixtures of odorants that have unfamiliar smells. The tests can be self-administered by healthy individuals with minimal training and show high test-retest reliability. Because SMELL-S uses odor mixtures rather than a single molecule, odor-specific insensitivity is averaged out, and the test accurately distinguished people with normal and dysfunctional smell. SMELL-R is a discrimination test in which the difference between two stimulus mixtures can be altered stepwise. This is an advance over current discrimination tests, which ask subjects to discriminate monomolecular odorants whose difference in odor cannot be quantified. SMELL-R showed significantly less bias in scores between North American and Taiwanese subjects than conventional semantically based smell tests that need to be adapted to different languages and cultures. Based on these proof-of-principle results in healthy individuals, we predict that SMELL-S and SMELL-R will be broadly effective in diagnosing smell dysfunction.

Predicting human olfactory perception from chemical features of odor molecules.

It is still not possible to predict whether a given molecule will have a perceived odor or what olfactory percept it will produce. We therefore organized the crowd-sourced DREAM Olfaction Prediction Challenge. Using a large olfactory psychophysical data set, teams developed machine-learning algorithms to predict sensory attributes of molecules based on their chemoinformatic features. The resulting models accurately predicted odor intensity and pleasantness and also successfully predicted 8 among 19 rated semantic descriptors ("garlic," "fish," "sweet," "fruit," "burnt," "spices," "flower," and "sour"). Regularized linear models performed nearly as well as random forest-based ones, with a predictive accuracy that closely approaches a key theoretical limit. These models help to predict the perceptual qualities of virtually any molecule with high accuracy and also reverse-engineer the smell of a molecule.