Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2. These mutants also fail to show CO2-evoked responses to heat and lactic acid, a human-derived attractant, suggesting that CO2 can gate responses to other sensory stimuli. Whereas attraction of Gr3 mutants to live humans in a large semi-field environment was only slightly impaired, responses to an animal host were greatly reduced in a spatial-scale-dependent manner. Synergistic integration of heat and odor cues likely drive host-seeking behavior in the absence of CO2 detection. We reveal a networked series of interactions by which multimodal integration of CO2, human odor, and heat orchestrates mosquito attraction to humans.

Targeting a dual detector of skin and CO2 to modify mosquito host seeking.

Female mosquitoes that transmit deadly diseases locate human hosts by detecting exhaled CO2 and skin odor. The identities of olfactory neurons and receptors required for attraction to skin odor remain a mystery. Here, we show that the CO2-sensitive olfactory neuron is also a sensitive detector of human skin odorants in both Aedes aegypti and Anopheles gambiae. We demonstrate that activity of this neuron is important for attraction to skin odor, establishing it as a key target for intervention. We screen ~0.5 million compounds in silico and identify several CO2 receptor ligands, including an antagonist that reduces attraction to skin and an agonist that lures mosquitoes to traps as effectively as CO2. Analysis of the CO2 receptor ligand space provides a foundation for understanding mosquito host-seeking behavior and identifies odors that are potentially safe, pleasant, and affordable for use in a new generation of mosquito control strategies worldwide.

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.

Reciprocal interactions between neuropeptide F and RYamide regulate host attraction in the mosquito Aedes aegypti.

Female mosquitoes produce eggs in gonadotrophic cycles that are divided between a previtellogenic and vitellogenic phase. Previtellogenic females consume water and sugar sources like nectar while also being attracted to hosts for blood feeding. Consumption of a blood meal activates the vitellogenic phase, which produces mature eggs and suppresses host attraction. In this study, we tested the hypothesis that neuropeptide Y-like hormones differentially modulate host attraction behavior in the mosquito Aedes aegypti. A series of experiments collectively indicated that enteroendocrine cells (EECs) in the posterior midgut produce and release neuropeptide F (NPF) into the hemolymph during the previtellogenic phase which stimulates attraction to humans and biting behavior. Consumption of a blood meal, which primarily consists of protein by dry weight, down-regulated NPF in EECs until mature eggs developed, which was associated with a decline in hemolymph titer. NPF depletion depended on protein digestion but was not associated with EEC loss. Other experiments showed that neurons in the terminal ganglion extend axons to the posterior midgut and produce RYamide, which showed evidence of increased secretion into circulation after a blood meal. Injection of RYamide-1 and -2 into previtellogenic females suppressed host attraction, while coinjection of RYamides with or without short NPF-2 also inhibited the host attraction activity of NPF. Overall, our results identify NPF and RYamide as gut-associated hormones in A. aegypti that link host attraction behavior to shifts in diet during sequential gonadotrophic cycles.

Incompatible and sterile insect techniques combined eliminate mosquitoes.

The radiation-based sterile insect technique (SIT) has successfully suppressed field populations of several insect pest species, but its effect on mosquito vector control has been limited. The related incompatible insect technique (IIT)-which uses sterilization caused by the maternally inherited endosymbiotic bacteria Wolbachia-is a promising alternative, but can be undermined by accidental release of females infected with the same Wolbachia strain as the released males. Here we show that combining incompatible and sterile insect techniques (IIT-SIT) enables near elimination of field populations of the world's most invasive mosquito species, Aedes albopictus. Millions of factory-reared adult males with an artificial triple-Wolbachia infection were released, with prior pupal irradiation of the released mosquitoes to prevent unintentionally released triply infected females from successfully reproducing in the field. This successful field trial demonstrates the feasibility of area-wide application of combined IIT-SIT for mosquito vector control.

Essential functions of mosquito ecdysone importers in development and reproduction.

The primary insect steroid hormone ecdysone requires a membrane transporter to enter its target cells. Although an organic anion-transporting polypeptide (OATP) named Ecdysone Importer (EcI) serves this role in the fruit fly Drosophila melanogaster and most likely in other arthropod species, this highly conserved transporter is apparently missing in mosquitoes. Here we report three additional OATPs that facilitate cellular incorporation of ecdysone in Drosophila and the yellow fever mosquito Aedes aegypti. These additional ecdysone importers (EcI-2, -3, and -4) are dispensable for development and reproduction in Drosophila, consistent with the predominant role of EcI. In contrast, in Aedes, EcI-2 is indispensable for ecdysone-mediated development, whereas EcI-4 is critical for vitellogenesis induced by ecdysone in adult females. Altogether, our results indicate unique and essential functions of these additional ecdysone importers in mosquito development and reproduction, making them attractive molecular targets for species- and stage-specific control of ecdysone signaling in mosquitoes.

Meeting a threat of the Anthropocene: Taste avoidance of metal ions by Drosophila.

The Anthropocene Epoch poses a critical challenge for organisms: they must cope with new threats at a rapid rate. These threats include toxic chemical compounds released into the environment by human activities. Here, we examine elevated concentrations of heavy metal ions as an example of anthropogenic stressors. We find that the fruit fly Drosophila avoids nine metal ions when present at elevated concentrations that the flies experienced rarely, if ever, until the Anthropocene. We characterize the avoidance of feeding and egg laying on metal ions, and we identify receptors, neurons, and taste organs that contribute to this avoidance. Different subsets of taste receptors, including members of both Ir (Ionotropic receptor) and Gr (Gustatory receptor) families contribute to the avoidance of different metal ions. We find that metal ions activate certain bitter-sensing neurons and inhibit sugar-sensing neurons. Some behavioral responses are mediated largely through neurons of the pharynx. Feeding avoidance remains stable over 10 generations of exposure to copper and zinc ions. Some responses to metal ions are conserved across diverse dipteran species, including the mosquito Aedes albopictus. Our results suggest mechanisms that may be essential to insects as they face challenges from environmental changes in the Anthropocene.

Juvenile hormone-induced histone deacetylase 3 suppresses apoptosis to maintain larval midgut in the yellow fever mosquito.

SignificanceJuvenile hormone (JH), a sesquiterpenoid, regulates many aspects of insect development, including maintenance of the larval stage by preventing metamorphosis. In contrast, ecdysteroids promote metamorphosis by inducing the E93 transcription factor, which triggers apoptosis of larval cells and remodeling of the larval midgut. We discovered that JH suppresses precocious larval midgut-remodeling by inducing an epigenetic modifier, histone deacetylase 3 (HDAC3). JH-induced HDAC3 deacetylates the histone H4 localized at the promoters of proapoptotic genes, resulting in the suppression of these genes. This eventually prevents programmed cell death of midgut cells and midgut-remodeling during larval stages. These studies identified a previously unknown mechanism of JH action in blocking premature remodeling of the midgut during larval feeding stages.

Dehydration stress and Mayaro virus vector competence in Aedes aegypti.

IMPORTANCE: Relative humidity (RH) is an environmental variable that affects mosquito physiology and can impact pathogen transmission. Low RH can induce dehydration in mosquitoes, leading to alterations in physiological and behavioral responses such as blood-feeding and host-seeking behavior. We evaluated the effects of a temporal drop in RH (RH shock) on mortality and Mayaro virus vector competence in Ae. aegypti. While dehydration induced by humidity shock did not impact virus infection, we detected a significant effect of dehydration on mosquito mortality and blood-feeding frequency, which could significantly impact transmission dynamics.

Population-specific responses to developmental temperature in the arboviral vector Aedes albopictus: Implications for climate change.

The increase of environmental temperature due to current global warming is not only favouring the expansion of the distribution range of many insect species, but it is also changing their phenology. Insect phenology is tightly linked to developmental timing, which is regulated by environmental temperatures. However, the degree to which the effects of developmental temperatures extend across developmental stages and their inter-stage relationships have not been thoroughly quantified in mosquitoes. Here, we used the mosquito Aedes albopictus, which is an aggressive invasive species and an arboviral vector, to study how developmental temperature influences fitness across developmental stages, thermal traits, energy reserves, transcriptome and Wolbachia prevalence in laboratory-reared populations originally collected from either temperate or tropical regions. We show that hatchability, larval and pupal viability and developmental speed are strongly influenced by temperature, and these effects extend to wing length, body mass, longevity and content of water, protein and lipids in adults in a population-specific manner. On the contrary, neither adult thermal preference nor heat resistance significantly change with temperature. Wolbachia density was generally lower in adult mosquitoes reared at 18°C than at other tested temperatures, and transcriptome analysis showed enrichment for functions linked to stress responses (i.e. cuticle proteins and chitin, cytochrome p450 and heat shock proteins) in mosquitoes reared at both 18 and 32°C. Our data showed an overall reduced vector fitness performance when mosquitoes were reared at 32°C, and the absence of isomorphy in the relationship between developmental stages and temperature in the laboratory population deriving from larvae collected in northern Italy. Altogether, these results have important implications for reliable model projections of the invasion potentials of Ae. albopictus and its epidemiological impact.

Phenotypic adaptation to temperature in the mosquito vector, Aedes aegypti.

Most models exploring the effects of climate change on mosquito-borne disease ignore thermal adaptation. However, if local adaptation leads to changes in mosquito thermal responses, "one size fits all" models could fail to capture current variation between populations and future adaptive responses to changes in temperature. Here, we assess phenotypic adaptation to temperature in Aedes aegypti, the primary vector of dengue, Zika, and chikungunya viruses. First, to explore whether there is any difference in existing thermal response of mosquitoes between populations, we used a thermal knockdown assay to examine five populations of Ae. aegypti collected from climatically diverse locations in Mexico, together with a long-standing laboratory strain. We identified significant phenotypic variation in thermal tolerance between populations. Next, to explore whether such variation can be generated by differences in temperature, we conducted an experimental passage study by establishing six replicate lines from a single field-derived population of Ae. aegypti from Mexico, maintaining half at 27°C and the other half at 31°C. After 10 generations, we found a significant difference in mosquito performance, with the lines maintained under elevated temperatures showing greater thermal tolerance. Moreover, these differences in thermal tolerance translated to shifts in the thermal performance curves for multiple life-history traits, leading to differences in overall fitness. Together, these novel findings provide compelling evidence that Ae. aegypti populations can and do differ in thermal response, suggesting that simplified thermal performance models might be insufficient for predicting the effects of climate on vector-borne disease transmission.

Voltage-gated ion channels are expressed in the Malpighian tubules and anal papillae of the yellow fever mosquito (Aedes aegypti), and may regulate ion transport during salt and water imbalance.

Vectors of infectious disease include several species of Aedes mosquitoes. The life cycle of Aedes aegypti, the yellow fever mosquito, consists of a terrestrial adult and an aquatic larval life stage. Developing in coastal waters can expose larvae to fluctuating salinity, causing salt and water imbalance, which is addressed by two prime osmoregulatory organs - the Malpighian tubules (MTs) and anal papillae (AP). Voltage-gated ion channels (VGICs) have recently been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects. In the current study, we: (i) generated MT transcriptomes of freshwater-acclimated and brackish water-exposed larvae of Ae. aegypti, (ii) detected expression of several voltage-gated Ca2+, K+, Na+ and non-ion-selective ion channels in the MTs and AP using transcriptomics, PCR and gel electrophoresis, (iii) demonstrated that mRNA abundance of many altered significantly following brackish water exposure, and (iv) immunolocalized CaV1, NALCN, TRP/Painless and KCNH8 in the MTs and AP of larvae using custom-made antibodies. We found CaV1 to be expressed in the apical membrane of MTs of both larvae and adults, and its inhibition to alter membrane potentials of this osmoregulatory epithelium. Our data demonstrate that multiple VGICs are expressed in osmoregulatory epithelia of Ae. aegypti and may play an important role in the autonomous regulation of ion transport.

Identifying villages and breeding habitats for dengue transmission in Thailand: insights from long-term larval surveys.

BACKGROUND: In Thailand, the Department of Disease Control (DDC) regularly performs visual larval surveys throughout the country to monitor dengue fever outbreaks. Since 2016, the DDC switched from a paper-based to a digital-based larval survey process. The significant amount of larval survey data collected digitally presents a valuable opportunity to precisely identify the villages and breeding habitats that are vulnerable to dengue transmission. METHODS: The study used digitally collected larval survey data from 2017 to 2019. It employed larval indices to evaluate the risk of dengue transmission in villages based on seasonal, regional, and categorical perspectives. Furthermore, the study comprehensively scrutinized each container category by employing different measures to determine its breeding preference ratio. RESULTS: The result showed that villages with a very high-risk of dengue transmission were present year-round in all regions, with the highest proportion during the rainy season. The Southern region had more high-risk villages during the winter season due to rainfall. Slums and residential communities were more vulnerable to dengue than commercial areas. All container categories could potentially serve as breeding habitats for dengue-carrying mosquitoes, with abandoned containers being the most significant breeding sites. CONCLUSIONS: The risk of dengue transmission was present year-round throughout Thailand. This underscores the importance of community and government initiatives, along with sustained public awareness campaigns and active community engagement, to efficiently and permanently eradicate mosquito breeding habitats. It should be noted that larval indices may not strongly correlate with dengue cases, as indicated by the preliminary analysis. However, they offer valuable insights into potential breeding sites for targeted preventive measures.

A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium.

Wolbachia are maternally inherited intracellular bacterial symbionts that are estimated to infect more than 60% of all insect species. While Wolbachia is commonly found in many mosquitoes it is absent from the species that are considered to be of major importance for the transmission of human pathogens. The successful introduction of a life-shortening strain of Wolbachia into the dengue vector Aedes aegypti that halves adult lifespan has recently been reported. Here we show that this same Wolbachia infection also directly inhibits the ability of a range of pathogens to infect this mosquito species. The effect is Wolbachia strain specific and relates to Wolbachia priming of the mosquito innate immune system and potentially competition for limiting cellular resources required for pathogen replication. We suggest that this Wolbachia-mediated pathogen interference may work synergistically with the life-shortening strategy proposed previously to provide a powerful approach for the control of insect transmitted diseases.

Mosquitoes do not Like Bitter.

Chemical repellents play a crucial role in personal protection, serving as essential elements in reducing the transmission of vector-borne diseases. A biorational perspective that extends beyond the olfactory system as the classical target may be a promising direction to move. The taste system provides reliable information regarding food quality, helping animals to discriminate between nutritious and potentially harmful food sources, often associated with a bitter taste. Understanding how bitter compounds affect feeding in blood-sucking insects could unveil novel molecules with the potential to reduce biting and feeding. Here, we investigated the impact of two naturally occurring bitter compounds, caffeine and quinine, on the feeding decisions in female Aedes aegypti mosquitoes at two distinctive phases: (1) when the mosquito explores the biting substrate using external taste sensors and (2) when the mosquito takes a sip of food and tastes it using internal taste receptors. We assessed the aversiveness of bitter compounds through both an artificial feeding condition (artificial feeder test) and a real host (arm-in-cage test). Our findings revealed different sensitivities in the external and internal sensory pathways responsible for detecting bitter taste in Ae. aegypti. Internal detectors exhibited responsiveness to lower doses compared to the external sensors. Quinine exerted a more pronounced negative impact on biting and feeding activity than caffeine. The implications of our findings are discussed in the context of mosquito food recognition and the potential practical implications for personal protection.

Assessment of Mayaro virus vector competence of the mosquito Aedes aegypti (Linnaeus, 1762) populations in Argentine using dose-response assays.

Mayaro virus (MAYV; Alphavirus: Togaviridae) is an emerging pathogen in Latin America, causing fever and polyarthritis. Sporadic outbreaks of MAYV have occurred in the region, with reported human cases being imported to Europe and North America. Although primarily a risk for those residing in the Amazon basin's tropical forests, recent reports highlight that urbanization would increase the risk of MAYV transmission in Latin America. Urban emergence depends on human susceptibility and the ability of mosquitos like Aedes aegypti  (Linnaeus, 1762) (Diptera: Culicidae) to transmit MAYV. Despite the absence of active MAYV transmission in Argentine, the risk of introduction is substantial due to human movement and the presence of Ae. aegypti in the region. This study aimed to evaluate the susceptibility of different Argentine Ae. aegypti populations to MAYV genotype L (MAYV-L) using dose-response assays and determine barriers to virus infection, dissemination and transmission. Immature mosquito stages were collected in Buenos Aires, Córdoba and Rosario cities. Female Ae. aegypti (F2) were orally infected by feeding on five concentrations of MAYV-L, ranging from 1.0 to 6.0 log10 PFU/mL. Abdomens, legs and saliva were analysed using viral plaque assays. Results revealed that MAYV-L between infection and dissemination were associated with viral doses rather than the population origin. Infection rates varied between 3% and 65%, with a 50% infectious dose >5.5 log10 PFU/mL. Dissemination occurred at 39%, with a 50% dissemination dose of ~6.0 log10 PFU/mL. Dissemination among infected mosquitoes ranged from 60% to 86%, and transmission from disseminated mosquitoes ranged from 11% to 20%. Argentine Ae. aegypti populations exhibited a need for higher viral doses of MAYV-L than those typically found in humans to become infected. In addition, only a small proportion of infected mosquitoes were capable of transmitting the virus. Understanding MAYV transmission in urban areas is crucial for public health interventions.

Application of computer vision and deep learning models to automatically classify medically important mosquitoes in North Borneo, Malaysia.

Mosquito-borne diseases have emerged in North Borneo in Malaysia due to rapid changes in the forest landscape, and mosquito surveillance is key to understanding disease transmission. However, surveillance programmes involving sampling and taxonomic identification require well-trained personnel, are time-consuming and labour-intensive. In this study, we aim to use a deep leaning model (DL) to develop an application capable of automatically detecting mosquito vectors collected from urban and suburban areas in North Borneo, Malaysia. Specifically, a DL model called MobileNetV2 was developed using a total of 4880 images of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus mosquitoes, which are widely distributed in Malaysia. More importantly, the model was deployed as an application that can be used in the field. The model was fine-tuned with hyperparameters of learning rate 0.0001, 0.0005, 0.001, 0.01 and the performance of the model was tested for accuracy, precision, recall and F1 score. Inference time was also considered during development to assess the feasibility of the model as an app in the real world. The model showed an accuracy of at least 97%, a precision of 96% and a recall of 97% on the test set. When used as an app in the field to detect mosquitoes with the elements of different background environments, the model was able to achieve an accuracy of 76% with an inference time of 47.33 ms. Our result demonstrates the practicality of computer vision and DL in the real world of vector and pest surveillance programmes. In the future, more image data and robust DL architecture can be explored to improve the prediction result.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Cross-talk of insulin-like peptides, juvenile hormone, and 20-hydroxyecdysone in regulation of metabolism in the mosquito Aedes aegypti.

Female mosquitoes feed sequentially on carbohydrates (nectar) and proteins (blood) during each gonadotrophic cycle to become reproductively competent and effective disease vectors. Accordingly, metabolism is synchronized to support this reproductive cyclicity. However, regulatory pathways linking metabolism to reproductive cycles are not fully understood. Two key hormones, juvenile hormone (JH) and ecdysteroids (20-hydroxyecdysone, 20E, is the most active form) govern female mosquito reproduction. Aedes aegypti genome codes for eight insulin-like peptides (ILPs) that are critical for controlling metabolism. We examined the effects of the JH and 20E pathways on mosquito ILP expression to decipher regulation of metabolism in a reproducing female mosquito. Chromatin immunoprecipitation assays showed genomic interactions between ilp genes and the JH receptor, methoprene-tolerant, a transcription factor, Krüppel homolog 1 (Kr-h1), and two isoforms of the ecdysone response early gene, E74. The luciferase reporter assays showed that Kr-h1 activates ilps 2, 6, and 7, but represses ilps 4 and 5 The 20E pathway displayed the opposite effect in the regulation of ilps E74B repressed ilps 2 and 6, while E74A activated ilps 4 and 5 Combining RNA interference, CRISPR gene tagging and enzyme-linked immunosorbent assay, we have shown that the JH and 20E regulate protein levels of all eight Ae. aegypti ILPs. Thus, we have established a regulatory axis between ILPs, JH, and 20E in coordination of metabolism during gonadotrophic cycles of Ae. aegypti.

The potential habitat and environmental fitness change of Aedes albopictus in Western Eurasia for 2081-2100.

BACKGROUND OBJECTIVES: The range of Aedes albopictus, the most important vector mosquito in Western Eurasia is growing due to climate change. However, it is not known how it will influence the habitats occupied by the species and its environmental fitness within its future range. METHODS: To study this question, the habitat characteristic of the mosquito was investigated for 2081-2100. RESULTS: The models suggest a notable future spread of the mosquito in the direction of Northern Europe and the parallel northward and westward shift of the southern and eastern potential occurrences of the mosquito. The models suggest a notable increase in generation numbers in the warmest quarter, which can reach 4-5 generations in the peri-Mediterranean region. However, both the joint survival rate of larvae and pupae and the number of survival days of adults in the warmest quarter exhibit decreasing values, as does the potential disappearance of the mosquito in the southern regions of Europe and Asia Minor, along with the growing atmospheric CO2 concentration-based scenarios. INTERPRETATION CONCLUSION: While in 1970-2000 Aedes albopictus mainly occupied the hot and warm summer temperate regions of Europe, the species will inhabit dominantly the cool summer temperate (oceanic) and the humid continental climate territories of North and North-Eastern Europe in 2081-2100.