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.

Sex peptide receptor is not required for refractoriness to remating or induction of egg laying in Aedes aegypti.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating-processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating postmating responses in a non-Drosophila dipteran, we generated 2 independent Spr-knockout alleles in the yellow fever mosquito, Aedes aegypti. Although SPR is needed for postmating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal postmating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these postmating responses. Our results demonstrate that Spr is not required for these canonical postmating responses in Ae. aegypti, indicating that other, as yet unknown, signaling pathways are likely responsible for these behavioral switches in this disease vector.

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.

Characterizing physical interactions between male and female mosquitoes (Aedes aegypti) in relation to female receptivity and insemination outcomes using a hydrophobic fluorescent dye.

Aedes aegypti, the yellow fever mosquito, presents a major threat to human health across the globe as a vector of disease-causing pathogens. Females of this species generally mate only once. From this single mating event, the female stores sufficient sperm to fertilize the multiple clutches of eggs produced during her lifetime. Mating causes dramatic changes in the female's behavior and physiology, including a lifetime suppression of her mating receptivity. Female rejection behaviors include male avoidance, abdominal twisting, wing-flicking, kicking, and not opening vaginal plates or extruding the ovipositor. Many of these events occur on a scale that is too miniscule or fast to see by eye, so high-resolution videography has been used to observe these behaviors instead. However, videography can be labor intensive, require specialized equipment, and often requires restrained animals. We used an efficient, low-cost method to record physical contact between males and females during attempted and successful mating, determined by recording spermathecal filling after dissection. A hydrophobic oil-based fluorescent dye can be applied to the abdominal tip of one animal and can be subsequently transferred to the genitalia of animals of the opposite sex when genital contact occurs. Our data indicate that male mosquitoes make high levels of contact with both receptive and unreceptive females and that males attempt to mate with more females than they successfully inseminate. Female mosquitoes with disrupted remating suppression mate with and produce offspring from multiple males, transferring dye to each. These data suggest that physical copulatory interactions occur independently of the female's receptivity to mate and that many of these interactions represent unsuccessful mating attempts that do not result in insemination.

Characterizing Physical Interactions between Male and Female Mosquitoes (Aedes aegypti) in Relation to Female Receptivity and Insemination Outcomes Using a Hydrophobic Fluorescent Dye.

Aedes aegypti, the yellow fever mosquito, presents a major threat to human health across the globe as a vector of disease-causing pathogens. Females of this species generally mate only once. From this single mating event, the female stores sufficient sperm to fertilize the multiple clutches of eggs produced during her lifetime. Mating causes dramatic changes in the female's behavior and physiology, including a lifetime suppression of her mating receptivity. Female rejection behaviors include male avoidance, abdominal twisting, wing-flicking, kicking, and not opening vaginal plates or extruding the ovipositor. Many of these events occur on a scale that is too miniscule or fast to see by eye, so high-resolution videography has been used to observe these behaviors instead. However, videography can be labor intensive, require specialized equipment, and often requires restrained animals. We used an efficient, low-cost method to record physical contact between males and females during attempted and successful mating, determined by recording spermathecal filling after dissection. A hydrophobic oil-based fluorescent dye can be applied to the abdominal tip of one animal and can be subsequently transferred to the genitalia of animals of the opposite sex when genital contact occurs. Our data indicate that male mosquitoes make high levels of contact with both receptive and unreceptive females and that males attempt to mate with more females than they successfully inseminate. Female mosquitoes with disrupted remating suppression mate with and produce offspring from multiple males, transferring dye to each. These data suggest that physical copulatory interactions occur independently of the female's receptivity to mate and that many of these interactions represent unsuccessful mating attempts that do not result in insemination.

Vector biology: A mosquito's deadly kiss on the LIPS.

A new study identifies a mosquito salivary protein that directly binds to a cuticular partner during biting to reshape the mosquito mouthparts, stimulate salivation and probing, and enhance blood-feeding efficiency. By affecting mosquito-host interactions, this phenomenon could influence pathogen transmission.

Maternally Instigated Diapause in Aedes albopictus: Coordinating Experience and Internal State for Survival in Variable Environments.

The Asian tiger mosquito, Aedes albopictus, is one of the most dangerous invasive species in the world. Females bite mammalian hosts, including humans, to obtain blood for egg development. The ancestral range of Ae. albopictus likely spanned from India to Japan and this species has since invaded a substantial portion of the globe. Ae. albopictus can be broadly categorized into temperate and tropical populations. One key to their ability to invade diverse ecological spaces is the capacity of females to detect seasonal changes and produce stress-resistant eggs that survive harsh winters. Females living in temperate regions respond to cues that predict the onset of unfavorable environmental conditions by producing eggs that enter maternally instigated embryonic diapause, a developmentally arrested state, which allows species survival by protecting the embryos until favorable conditions return. To appropriately produce diapause eggs, the female must integrate environmental cues and internal physiological state (blood feeding and reproductive status) to allocate nutrients and regulate reproduction. There is variation in reproductive responses to environmental cues between interfertile tropical and temperate populations depending on whether females are actively producing diapause vs. non-diapause eggs and whether they originate from populations that are capable of diapause. Although diapause-inducing environmental cues and diapause eggs have been extensively characterized, little is known about how the female detects gradual environmental changes and coordinates her reproductive status with seasonal dynamics to lay diapause eggs in order to maximize offspring survival. Previous studies suggest that the circadian system is involved in detecting daylength as a critical cue. However, it is unknown which clock network components are important, how these connect to reproductive physiology, and how they may differ between behavioral states or across populations with variable diapause competence. In this review, we showcase Ae. albopictus as an emerging species for neurogenetics to study how the nervous system combines environmental conditions and internal state to optimize reproductive behavior. We review environmental cues for diapause induction, downstream pathways that control female metabolic changes and reproductive capacity, as well as diapause heterogeneity between populations with different evolutionary histories. We highlight genetic tools that can be implemented in Ae. albopictus to identify signaling molecules and cellular circuits that control diapause. The tools and discoveries made in this species could translate to a broader understanding of how environmental cues are interpreted to alter reproductive physiology in other species and how populations with similar genetic and circuit organizations diversify behavioral patterns. These approaches may yield new targets to interfere with mosquito reproductive capacity, which could be exploited to reduce mosquito populations and the burden of the pathogens they transmit.

Size Quantification of Blood and Sugar Meals in Aedes aegypti Mosquitoes.

Both male and female mosquitoes consume sugar-rich nectar meals required for metabolic energy, but only females consume protein-rich blood meals, which are required for egg development. The size of each meal consumed has subsequent effects on behavior and reproduction; therefore, precise quantification is an important aspect of mosquito feeding behavior studies. This protocol describes a high-throughput, end-point assay to quantify meal volumes ingested by individual mosquitoes. The addition of a fluorescent dye to the meal allows for meal size quantification. Individual mosquitoes that have been fed this meal are homogenized in 96-well plates, and the fluorescence levels are measured with a plate reader. This protocol can also be adapted to determine if alteration of meal composition affects the ingested meal volume, if mosquito strain or genotype dictates consumption, or if meals are derived from multiple sources.

Methods to Assess Blood and Nectar Meals in Aedes aegypti Mosquitoes.

Male and female Aedes aegypti mosquitoes survive by feeding on floral nectar for metabolic energy, but females require blood protein, obtained from biting a host, for egg development. Although males exclusively derive energy from nectar sugars, females must select the meal that best matches their present metabolic and reproductive needs. In females, blood and nectar promote independent feeding behaviors with distinct sensory appendages, meal sizes, digestive tract targets, and metabolic fates. Understanding how male and female mosquitoes recognize, locate, and metabolize nutrients is essential for characterizing the survival and reproductive capabilities of this mosquito. Here, we provide an introduction to blood versus nectar feeding and methods to quantify nectar and blood meal sizes in individual Ae. aegypti mosquitoes. Precise quantification of meal size is crucial for ensuring consistency in assays that record events downstream of feeding behavior, including host attraction or fecundity.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes.

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females. Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito. These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Mosquito Host-Seeking Regulation: Targets for Behavioral Control.

Female Aedes aegypti mosquitoes require protein from blood to develop eggs. They have evolved a strong innate drive to find and bite humans and engorge on their blood. Decades of research have revealed that attraction to hosts is suppressed for days after blood-feeding. During this time, females coordinate complex physiological changes, allowing them to utilize blood protein to develop eggs: clearing excess fluid, digesting protein, and egg maturation. How do mechanosensation, nutrient consumption, and reproductive pathways combine to produce the full expression of host-seeking suppression? Understanding mechanisms of endogenous host-seeking suppression may allow them to be 'weaponized' against mosquitoes through exogenous activation and developed as tools for vector control. Recent work allows unprecedented genetic and pharmacological access to characterize and disrupt this behavioral cycle.

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.

E and M circadian pacemaker neurons use different PDF receptor signalosome components in drosophila.

We used real-time imaging to detect cAMP levels in neurons of intact fly brains to study the mechanisms of circadian pacemaker synchronization by the neuropeptide pigment dispersing factor (PDF) in Drosophila. PDF receptor (PDF-R) is expressed by both M (sLNv) and E (LNd) pacemaker subclasses and is coupled to G(sα) in both cases. We previously reported that PDF-R in M pacemakers elevates cAMP levels by activating the ortholog of mammalian adenylate cyclase 3 (AC3) but that AC3 disruptions had no effect on E pacemaker sensitivity to PDF. Here, we show that PDF-R in E pacemakers activates a different AC isoform, AC78C, an ortholog of mammalian AC8. Knockdown of AC78C by transgenic RNAi substantially reduces, but does not completely abrogate, PDF responses in these E pacemakers. The knockdown effect is intact when restricted to mature stages, suggesting a physiological and not a development role for AC78C in E pacemakers. The AC78C phenotype is rescued by the overexpression of AC78C but not by overexpression of the rutabaga AC. AC78C overexpression does not disrupt PDF responses in these E pacemakers, and neither AC78C knockdown nor its overexpression disrupted locomotor rhythms. Finally, knockdown of 2 AKAPs, nervy and AKAP200, partially reduces LNd PDF responses. These findings begin to identify the components of E pacemaker PDF-R signalosomes and indicate that they are distinct from PDF-R signalosomes in M pacemakers: we propose they contain AC78C and at least 1 other AC.

The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

Circadian rhythms: biological clocks work in phospho-time.

The 24 hour molecular oscillator requires precisely calibrated degradation of core clock proteins, like PERIOD. New studies shed light on a sequential series of PERIOD phosphorylation events that first inhibits, then accelerates PERIOD degradation.