The Perfect Appetizer: A Pharmacological Strategy for a Non-biting Mosquito.

New possibilities for vector-borne disease control are revealed by Duvall et al. (2019), who link host-seeking behavioral modulation in Aedes aegypti to neuropeptide Y (NPY)-like receptor 7. Small-molecule screening yields agonist compounds able to activate NPYLR7 and suppress attraction to hosts.

Steroid Hormone Function Controls Non-competitive Plasmodium Development in Anopheles.

Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

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.

Salivary factor LTRIN from Aedes aegypti facilitates the transmission of Zika virus by interfering with the lymphotoxin-ß receptor.

Pathogens have co-evolved with mosquitoes to optimize transmission to hosts. Mosquito salivary-gland extract is known to modulate host immune responses and facilitate pathogen transmission, but the underlying molecular mechanisms of this have remained unknown. In this study, we identified and characterized a prominent 15-kilodalton protein, LTRIN, obtained from the salivary glands of the mosquito Aedes aegypti. LTRIN expression was upregulated in blood-fed mosquitoes, and LTRIN facilitated the transmission of Zika virus (ZIKV) and exacerbated its pathogenicity by interfering with signaling through the lymphotoxin-ß receptor (LTßR). Mechanically, LTRIN bound to LTßR and 'preferentially' inhibited signaling via the transcription factor NF-κB and the production of inflammatory cytokines by interfering with the dimerization of LTßR during infection with ZIKV. Furthermore, treatment with antibody to LTRIN inhibited mosquito-mediated infection with ZIKV, and abolishing LTßR potentiated the infectivity of ZIKV both in vitro and in vivo. This study provides deeper insight into the transmission of mosquito-borne diseases in nature and supports the therapeutic potential of inhibiting the action of LTRIN to disrupt ZIKV transmission.

A mosquito salivary protein-driven influx of myeloid cells facilitates flavivirus transmission.

Mosquitoes transmit many disease-relevant flaviviruses. Efficient viral transmission to mammalian hosts requires mosquito salivary factors. However, the specific salivary components facilitating viral transmission and their mechanisms of action remain largely unknown. Here, we show that a female mosquito salivary gland-specific protein, here named A. aegypti Neutrophil Recruitment Protein (AaNRP), facilitates the transmission of Zika and dengue viruses. AaNRP promotes a rapid influx of neutrophils, followed by virus-susceptible myeloid cells toward mosquito bite sites, which facilitates establishment of local infection and systemic dissemination. Mechanistically, AaNRP engages TLR1 and TLR4 of skin-resident macrophages and activates MyD88-dependent NF-κB signaling to induce the expression of neutrophil chemoattractants. Inhibition of MyD88-NF-κB signaling with the dietary phytochemical resveratrol reduces AaNRP-mediated enhancement of flavivirus transmission by mosquitoes. These findings exemplify how salivary components can aid viral transmission, and suggest a potential prophylactic target.

VLDLR and ApoER2 are receptors for multiple alphaviruses.

Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1-3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2-E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD-Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2-E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD-Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.

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.

A male steroid controls female sexual behaviour in the malaria mosquito.

Insects, unlike vertebrates, are widely believed to lack male-biased sex steroid hormones1. In the malaria mosquito Anopheles gambiae, the ecdysteroid 20-hydroxyecdysone (20E) appears to have evolved to both control egg development when synthesized by females2 and to induce mating refractoriness when sexually transferred by males3. Because egg development and mating are essential reproductive traits, understanding how Anopheles females integrate these hormonal signals can spur the design of new malaria control programs. Here we reveal that these reproductive functions are regulated by distinct sex steroids through a sophisticated network of ecdysteroid-activating/inactivating enzymes. We identify a male-specific oxidized ecdysteroid, 3-dehydro-20E (3D20E), which safeguards paternity by turning off female sexual receptivity following its sexual transfer and activation by dephosphorylation. Notably, 3D20E transfer also induces expression of a reproductive gene that preserves egg development during Plasmodium infection, ensuring fitness of infected females. Female-derived 20E does not trigger sexual refractoriness but instead licenses oviposition in mated individuals once a 20E-inhibiting kinase is repressed. Identifying this male-specific insect steroid hormone and its roles in regulating female sexual receptivity, fertility and interactions with Plasmodium parasites suggests the possibility for reducing the reproductive success of malaria-transmitting mosquitoes.

Evolutionary Ecology of Wolbachia Releases for Disease Control.

Wolbachia is an endosymbiotic Alphaproteobacteria that can suppress insect-borne diseases through decreasing host virus transmission (population replacement) or through decreasing host population density (population suppression). We contrast natural Wolbachia infections in insect populations with Wolbachia transinfections in mosquitoes to gain insights into factors potentially affecting the long-term success of Wolbachia releases. Natural Wolbachia infections can spread rapidly, whereas the slow spread of transinfections is governed by deleterious effects on host fitness and demographic factors. Cytoplasmic incompatibility (CI) generated by Wolbachia is central to both population replacement and suppression programs, but CI in nature can be variable and evolve, as can Wolbachia fitness effects and virus blocking. Wolbachia spread is also influenced by environmental factors that decrease Wolbachia titer and reduce maternal Wolbachia transmission frequency. More information is needed on the interactions between Wolbachia and host nuclear/mitochondrial genomes, the interaction between invasion success and local ecological factors, and the long-term stability of Wolbachia-mediated virus blocking.

Division and Transmission: Malaria Parasite Development in the Mosquito.

The malaria parasite life cycle alternates between two hosts: a vertebrate and the female Anopheles mosquito vector. Cell division, proliferation, and invasion are essential for parasite development, transmission, and survival. Most research has focused on Plasmodium development in the vertebrate, which causes disease; however, knowledge of malaria parasite development in the mosquito (the sexual and transmission stages) is now rapidly accumulating, gathered largely through investigation of the rodent malaria model, with Plasmodium berghei. In this review, we discuss the seminal genome-wide screens that have uncovered key regulators of cell proliferation, invasion, and transmission during Plasmodium sexual development. Our focus is on the roles of transcription factors, reversible protein phosphorylation, and molecular motors. We also emphasize the still-unanswered important questions around key pathways in cell division during the vector transmission stages and how they may be targeted in future studies.

Intra-lineage microevolution of Wolbachia leads to the emergence of new cytoplasmic incompatibility patterns.

Mosquitoes of the Culex pipiens complex are worldwide vectors of arbovirus, filarial nematodes, and avian malaria agents. In these hosts, the endosymbiotic bacteria Wolbachia induce cytoplasmic incompatibility (CI), i.e., reduced embryo viability in so-called incompatible crosses. Wolbachia infecting Culex pipiens (wPip) cause CI patterns of unparalleled complexity, associated with the amplification and diversification of cidA and cidB genes, with up to 6 different gene copies described in a single wPip genome. In wPip, CI is thought to function as a toxin-antidote (TA) system where compatibility relies on having the right antidotes (CidA) in the female to bind and neutralize the male's toxins (CidB). By repeating crosses between Culex isofemale lines over a 17 years period, we documented the emergence of a new compatibility type in real time and linked it to a change in cid genes genotype. We showed that loss of specific cidA gene copies in some wPip genomes results in a loss of compatibility. More precisely, we found that this lost antidote had an original sequence at its binding interface, corresponding to the original sequence at the toxin's binding interface. We showed that these original cid variants are recombinant, supporting a role for recombination rather than point mutations in rapid CI evolution. These results strongly support the TA model in natura, adding to all previous data acquired with transgenes expression.

The mechanism of cytoplasmic incompatibility is conserved in Wolbachia-infected Aedes aegypti mosquitoes deployed for arbovirus control.

The rising interest and success in deploying inherited microorganisms and cytoplasmic incompatibility (CI) for vector control strategies necessitate an explanation of the CI mechanism. Wolbachia-induced CI manifests in the form of embryonic lethality when sperm from Wolbachia-bearing testes fertilize eggs from uninfected females. Embryos from infected females however survive to sustain the maternally inherited symbiont. Previously in Drosophila melanogaster flies, we demonstrated that CI modifies chromatin integrity in developing sperm to bestow the embryonic lethality. Here, we validate these findings using wMel-transinfected Aedes aegypti mosquitoes released to control vector-borne diseases. Once again, the prophage WO CI proteins, CifA and CifB, target male gametic nuclei to modify chromatin integrity via an aberrant histone-to-protamine transition. Cifs are not detected in the embryo, and thus elicit CI via the nucleoprotein modifications established pre-fertilization. The rescue protein CifA in oogenesis localizes to stem cell, nurse cell, and oocyte nuclei, as well as embryonic DNA during embryogenesis. Discovery of the nuclear targeting Cifs and altered histone-to-protamine transition in both Aedes aegypti mosquitoes and D. melanogaster flies affirm the Host Modification Model of CI is conserved across these host species. The study also newly uncovers the cell biology of Cif proteins in the ovaries, CifA localization in the embryos, and an impaired histone-to-protamine transition during spermiogenesis of any mosquito species. Overall, these sperm modification findings may enable future optimization of CI efficacy in vectors or pests that are refractory to Wolbachia transinfections.

Advances and opportunities in malaria population genomics.

Almost 20 years have passed since the first reference genome assemblies were published for Plasmodium falciparum, the deadliest malaria parasite, and Anopheles gambiae, the most important mosquito vector of malaria in sub-Saharan Africa. Reference genomes now exist for all human malaria parasites and nearly half of the ~40 important vectors around the world. As a foundation for genetic diversity studies, these reference genomes have helped advance our understanding of basic disease biology and drug and insecticide resistance, and have informed vaccine development efforts. Population genomic data are increasingly being used to guide our understanding of malaria epidemiology, for example by assessing connectivity between populations and the efficacy of parasite and vector interventions. The potential value of these applications to malaria control strategies, together with the increasing diversity of genomic data types and contexts in which data are being generated, raise both opportunities and challenges in the field. This Review discusses advances in malaria genomics and explores how population genomic data could be harnessed to further support global disease control efforts.

Eliminating malaria vectors with precision-guided sterile males.

Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Geospatial joint modeling of vector and parasite serology to microstratify malaria transmission.

The World Health Organization identifies a strong surveillance system for malaria and its mosquito vector as an essential pillar of the malaria elimination agenda. Anopheles salivary antibodies are emerging biomarkers of exposure to mosquito bites that potentially overcome sensitivity and logistical constraints of traditional entomological surveys. Using samples collected by a village health volunteer network in 104 villages in Southeast Myanmar during routine surveillance, the present study employs a Bayesian geostatistical modeling framework, incorporating climatic and environmental variables together with Anopheles salivary antigen serology, to generate spatially continuous predictive maps of Anopheles biting exposure. Our maps quantify fine-scale spatial and temporal heterogeneity in Anopheles salivary antibody seroprevalence (ranging from 9 to 99%) that serves as a proxy of exposure to Anopheles bites and advances current static maps of only Anopheles occurrence. We also developed an innovative framework to perform surveillance of malaria transmission. By incorporating antibodies against the vector and the transmissible form of malaria (sporozoite) in a joint Bayesian geostatistical model, we predict several foci of ongoing transmission. In our study, we demonstrate that antibodies specific for Anopheles salivary and sporozoite antigens are a logistically feasible metric with which to quantify and characterize heterogeneity in exposure to vector bites and malaria transmission. These approaches could readily be scaled up into existing village health volunteer surveillance networks to identify foci of residual malaria transmission, which could be targeted with supplementary interventions to accelerate progress toward elimination.

Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter.

Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

Pathogen prospecting of museums: Reconstructing malaria epidemiology.

Malaria is a disease of global significance. Ongoing changes to the earth's climate, antimalarial resistance, insecticide resistance, and socioeconomic decline test the resilience of malaria prevention programs. Museum insect specimens present an untapped resource for studying vector-borne pathogens, spurring the question: Do historical mosquito collections contain Plasmodium DNA, and, if so, can museum specimens be used to reconstruct the historical epidemiology of malaria? In this Perspective, we explore molecular techniques practical to pathogen prospecting, which, more broadly, we define as the science of screening entomological museum specimens for human, animal, or plant pathogens. Historical DNA and pathogen prospecting provide a means of describing the coevolution of human, vector, and parasite, informing the development of insecticides, diagnostics, therapeutics, and vaccines.

Ecdysone-controlled nuclear receptor ERR regulates metabolic homeostasis in the disease vector mosquito Aedes aegypti.

Hematophagous mosquitoes require vertebrate blood for their reproductive cycles, making them effective vectors for transmitting dangerous human diseases. Thus, high-intensity metabolism is needed to support reproductive events of female mosquitoes. However, the regulatory mechanism linking metabolism and reproduction in mosquitoes remains largely unclear. In this study, we found that the expression of estrogen-related receptor (ERR), a nuclear receptor, is activated by the direct binding of 20-hydroxyecdysone (20E) and ecdysone receptor (EcR) to the ecdysone response element (EcRE) in the ERR promoter region during the gonadotropic cycle of Aedes aegypti (named AaERR). RNA interference (RNAi) of AaERR in female mosquitoes led to delayed development of ovaries. mRNA abundance of genes encoding key enzymes involved in carbohydrate metabolism (CM)-glucose-6-phosphate isomerase (GPI) and pyruvate kinase (PYK)-was significantly decreased in AaERR knockdown mosquitoes, while the levels of metabolites, such as glycogen, glucose, and trehalose, were elevated. The expression of fatty acid synthase (FAS) was notably downregulated, and lipid accumulation was reduced in response to AaERR depletion. Dual luciferase reporter assays and electrophoretic mobility shift assays (EMSA) determined that AaERR directly activated the expression of metabolic genes, such as GPI, PYK, and FAS, by binding to the corresponding AaERR-responsive motif in the promoter region of these genes. Our results have revealed an important role of AaERR in the regulation of metabolism during mosquito reproduction and offer a novel target for mosquito control.

Precise coordination between nutrient transporters ensures fertility in the malaria mosquito Anopheles gambiae.

Females from many mosquito species feed on blood to acquire nutrients for egg development. The oogenetic cycle has been characterized in the arboviral vector Aedes aegypti, where after a bloodmeal, the lipid transporter lipophorin (Lp) shuttles lipids from the midgut and fat body to the ovaries, and a yolk precursor protein, vitellogenin (Vg), is deposited into the oocyte by receptor-mediated endocytosis. Our understanding of how the roles of these two nutrient transporters are mutually coordinated is however limited in this and other mosquito species. Here, we demonstrate that in the malaria mosquito Anopheles gambiae, Lp and Vg are reciprocally regulated in a timely manner to optimize egg development and ensure fertility. Defective lipid transport via Lp knockdown triggers abortive ovarian follicle development, leading to misregulation of Vg and aberrant yolk granules. Conversely, depletion of Vg causes an upregulation of Lp in the fat body in a manner that appears to be at least partially dependent on target of rapamycin (TOR) signaling, resulting in excess lipid accumulation in the developing follicles. Embryos deposited by Vg-depleted mothers are completely inviable, and are arrested early during development, likely due to severely reduced amino acid levels and protein synthesis. Our findings demonstrate that the mutual regulation of these two nutrient transporters is essential to safeguard fertility by ensuring correct nutrient balance in the developing oocyte, and validate Vg and Lp as two potential candidates for mosquito control.

Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity.

Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infected Aedes aegypti mosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individual Ae. aegypti mosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles.