Multiple bouts of blood feeding in mosquitoes allow prolonged survival and are predicted to increase viral transmission during drought.

Survival through periods of drought is critical for mosquitoes to reside in semi-arid regions with humans, but water sources may be limited. Previous studies have shown that dehydrated mosquitoes will increase blood feeding propensity, but how this would occur over extended dry periods is unknown. Following a bloodmeal, prolonged exposure to dry conditions increased secondary blood feeding in mosquitoes by nearly two-fold, and chronic blood feeding allowed mosquitoes to survive twenty days without access to water sources. This refeeding did not alter the number of eggs generated, suggesting this refeeding is for hydration and nutrient replenishment. Exposure to desiccating conditions following a bloodmeal resulted in increased activity, decreased sleep levels, and prompted a return of CO2 sensing before egg deposition. The increased blood feeding during the vitellogenic stage and higher survival during dry periods are predicted to increase pathogen transmission and explain the elevated levels of specific arbovirus cases during dry conditions. These results solidify our understanding of the role of dry periods on mosquito blood feeding and how mosquito dehydration contributes to vectorial capacity and disease transmission dynamics.

Landscape and meteorological variables associated with Aedes aegypti and Aedes albopictus mosquito infestation in two southeastern USA coastal cities.

Aedes transmitted arboviral human cases are increasing worldwide and spreading to new areas of the United States of America (USA). These diseases continue to re-emerge likely due to changes in vector ecology, urbanization, human migration, and larger range of climatic suitability. Recent shifts in landscape and weather variables are predicted to impact the habitat patterns of urban mosquitoes such as Aedes aegypti and Aedes albopictus. Miami (FL) is in the tropical zone and an established hotspot for arboviruses, while Charleston (SC) is in the humid subtropical zone and newly vulnerable. Although these coastal cities have distinct climates, both have hot summers. To understand mosquito infestation in both cities and potentiate our surveillance effort, we performed egg collections in the warmest season. We applied remote sensing with land-use cover and weather variation to identify mosquito infestation patterns. Our study found predominant occurrence of Ae. aegypti and, to a lesser extent, Ae. albopictus in both cities. We detected statistically significant positive and negative associations between entomological indicators and most weather variables in combined data from both cities. For all entomological indices, weekly wind speed and relative humidity were significantly positively associated, while precipitation and maximum temperature were significantly negatively associated. Aedes egg abundance was significantly positively associated with open land in Charleston but was negatively associated with vegetation cover in combined data. There is a clear need for further observational studies to determine the impact of climate change on Ae. aegypti and Ae. albopictus infestation in the Southeastern region of the USA.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Fertility decline in Aedes aegypti (Diptera: Culicidae) mosquitoes is associated with reduced maternal transcript deposition and does not depend on female age.

Female mosquitoes undergo multiple rounds of reproduction known as gonotrophic cycles (GC). A gonotrophic cycle spans the period from blood meal intake to egg laying. Nutrients from vertebrate host blood are necessary for completing egg development. During oogenesis, a female prepackages mRNA into her oocytes, and these maternal transcripts drive the first 2 h of embryonic development prior to zygotic genome activation. In this study, we profiled transcriptional changes in 1-2 h of Aedes aegypti (Diptera: Culicidae) embryos across 2 GC. We found that homeotic genes which are regulators of embryogenesis are downregulated in embryos from the second gonotrophic cycle. Interestingly, embryos produced by Ae. aegypti females progressively reduced their ability to hatch as the number of GC increased. We show that this fertility decline is due to increased reproductive output and not the mosquitoes' age. Moreover, we found a similar decline in fertility and fecundity across 3 GC in Aedes albopictus. Our results are useful for predicting mosquito population dynamics to inform vector control efforts.

The pharyngeal taste organ of a blood-feeding insect functions in food recognition.

BACKGROUND: Obligate blood-feeding insects obtain the nutrients and water necessary to ensure survival from the vertebrate blood. The internal taste sensilla, situated in the pharynx, evaluate the suitability of the ingested food. Here, through multiple approaches, we characterized the pharyngeal organ (PO) of the hematophagous kissing bug Rhodnius prolixus to determine its role in food assessment. The PO, located antero-dorsally in the pharynx, comprises eight taste sensilla that become bathed with the incoming blood. RESULTS: We showed that these taste sensilla house gustatory receptor neurons projecting their axons through the labral nerves to reach the subesophageal zone in the brain. We found that these neurons are electrically activated by relevant appetitive and aversive gustatory stimuli such as NaCl, ATP, and caffeine. Using RNA-Seq, we examined the expression of sensory-related gene families in the PO. We identified gustatory receptors, ionotropic receptors, transient receptor potential channels, pickpocket channels, opsins, takeouts, neuropeptide precursors, neuropeptide receptors, and biogenic amine receptors. RNA interference assays demonstrated that the salt-related pickpocket channel Rproppk014276 is required during feeding of an appetitive solution of NaCl and ATP. CONCLUSIONS: We provide evidence of the role of the pharyngeal organ in food evaluation. This work shows a comprehensive characterization of a pharyngeal taste organ in a hematophagous insect.

Female Aedes aegypti mosquitoes use communal cues to manage population density at breeding sites.

Where a female mosquito lays her eggs creates the conditions for reproductive success. Here we identify a communal behavior among ovipositing female mosquitoes. When choosing equal breeding sites, gravid Aedes aegypti aggregate more often than expected. This aggregation occurs when water contact is restricted and does not require the presence of eggs. Instead, the aggregation is regulated by the number of females present at the breeding site. Using assays with both occupied and empty oviposition sites, we show that the Orco olfactory co-receptor and a carbon dioxide receptor, Gr3, detect the presence of mosquitoes. orco mutants aggregate more often in empty sites, suggesting attractive olfactory cues influence females to associate with one another. Gr3 mutant females do not prefer either site, suggesting that the CO2 receptor is necessary to evaluate mosquito population density at breeding sites. Further, raising CO2 levels is sufficient to cause wild-type mosquitoes to avoid empty oviposition sites. Our results demonstrate that female mosquitoes can regulate their own population density at breeding sites using attractive and repellent communal cues.

Odor-evoked transcriptomics of Aedes aegypti mosquitoes.

Modulation of odorant receptors mRNA induced by prolonged odor exposure is highly correlated with ligand-receptor interactions in Drosophila as well as mammals of the Muridae family. If this response feature is conserved in other organisms, this presents an intriguing initial screening tool when searching for novel receptor-ligand interactions in species with predominantly orphan olfactory receptors. We demonstrate that mRNA modulation in response to 1-octen-3-ol odor exposure occurs in a time- and concentration-dependent manner in Aedes aegypti mosquitoes. To investigate gene expression patterns at a global level, we generated an odor-evoked transcriptome associated with 1-octen-3-ol odor exposure. Transcriptomic data revealed that ORs and OBPs were transcriptionally responsive whereas other chemosensory gene families showed little to no differential expression. Alongside chemosensory gene expression changes, transcriptomic analysis found that prolonged exposure to 1-octen-3-ol modulated xenobiotic response genes, primarily members of the cytochrome P450, insect cuticle proteins, and glucuronosyltransferases families. Together, these findings suggest that mRNA transcriptional modulation of olfactory receptors caused by prolonged odor exposure is pervasive across taxa and can be accompanied by the activation of xenobiotic responses.

Carboxylic acids that drive mosquito attraction to humans activate ionotropic receptors.

The mosquito, Aedes aegypti, is highly anthropophilic and transmits debilitating arboviruses within human populations and between humans and non-human primates. Female mosquitoes are attracted to sources of blood by responding to odor plumes that are emitted by their preferred hosts. Acidic volatile compounds, including carboxylic acids, represent particularly salient odors driving this attraction. Importantly, carboxylic acids are major constituents of human sweat and volatiles generated by skin microbes. As such, they are likely to impact human host preference, a dominant factor in disease transmission cycles. A more complete understanding of mosquito host attraction will necessitate the elucidation of molecular mechanisms of volatile odor detection that function in peripheral sensory neurons. Recent studies have shown that members of the variant ionotropic glutamate receptor gene family are necessary for physiological and behavioral responses to acidic volatiles in Aedes. In this study, we have identified a subfamily of variant ionotropic receptors that share sequence homology across several important vector species and are likely to be activated by carboxylic acids. Moreover, we demonstrate that selected members of this subfamily are activated by short-chain carboxylic acids in a heterologous cell expression system. Our results are consistent with the hypothesis that members of this receptor class underlie acidic volatile sensitivity in vector mosquitoes and provide a frame of reference for future development of novel mosquito attractant and repellent technologies.

Fertility decline in female mosquitoes is regulated by the orco olfactory co-receptor.

Female Aedes aegypti mosquitoes undergo multiple rounds of reproduction, known as gonotrophic cycles. These cycles span the period from blood meal intake to oviposition. Understanding how reproductive success is maintained across gonotrophic cycles allows for the identification of molecular targets to reduce mosquito population growth. Odorant receptor co-receptor (orco) encodes a conserved insect-specific transmembrane ion channel that complexes with tuning odorant receptors (ORs) to form a functional olfactory receptor. orco expression has been identified in the male and female mosquito germline, but its role is unclear. We report an orco-dependent, maternal effect reduction in fertility after the first gonotrophic cycle. This phenotype was removed by CRISPR-Cas9 reversion of the orco mutant locus. Eggs deposited by orco mutant females are fertilized but the embryos reveal developmental defects, reduced hatching, and changes in ion channel signaling gene transcription. We present an unexpected role for an olfactory receptor pathway in mosquito reproduction.

Quantifying Mosquito Attraction Behavior Using Olfactometry.

When blood feeding from human hosts, female mosquitoes can transmit life-threatening pathogens to humans, including dengue virus, chikungunya virus, and Zika virus. Olfaction is the primary sense mosquitoes use to locate and differentiate hosts and studying it can lead to new strategies to reduce the risk of disease. To effectively study host-seeking behavior in mosquitoes, a repeatable, quantitative assay that isolates olfaction from other cues is critical for interpreting mosquito behavior. Here, we contribute an overview of methods and best practices for the study of mosquito attraction (or lack thereof) by using olfactometry to quantify behavior. In the accompanying protocols, we present an olfactory-based behavioral assay using a uniport olfactometer that measures mosquito attraction rate to specific stimuli. We include construction details, setup of the uniport olfactometer, details of the behavioral assay, and data analysis guidelines, as well as how to prepare the mosquitoes before their introduction into the olfactometer. This uniport olfactometer behavioral assay is currently one of the most reliable methods to study mosquito attraction to a single olfactory stimulus.

Building a Uniport Olfactometer to Assess Mosquito Responses to Odors.

The uniport olfactometer behavioral assay is currently one of the most reliable single-choice methods to use to study mosquito attraction to olfactory stimuli. It allows for the reproducible calculation of mosquito attraction rate to human hosts or to other olfactory stimuli. Here, we present the design of our modified uniport olfactometer. Consistent carbon-filtered air flows through the assay, creating positive pressure that reduces odor contamination from the room. It includes a precision-milled white acrylic base to facilitate easy setup and consistent placement of the component parts. Our design can be made by a commercial acrylic fabricator or an academic machine shop. This olfactometer is designed to assess the responses of mosquitoes but could be applied to other insects that fly upwind toward an odor stimulus. In a companion protocol, we detail how to perform the experiments with mosquitoes by using the uniport olfactometer.

Quantifying Mosquito Attraction Using a Uniport Olfactometer.

Female mosquitoes respond to the world around them by using chemosensory organs, such as their antennae, to detect volatile compounds emitted from a vertebrate host. These chemosensory systems facilitate the interpretation of external stimuli from the periphery by connecting to the central nervous system and eliciting behaviors necessary for survival, such as obtaining a blood meal. This innate behavior leads to the transmission of pathogens, including dengue virus, chikungunya virus, and Zika virus. Olfaction is a primary sense mosquitoes use to differentiate between vertebrate hosts, and studying it can lead to novel strategies to reduce the risk of disease. In this protocol, we present an olfactory-driven behavioral assay using a uniport olfactometer that measures mosquito attraction rate to a specific stimulus. We include details of the behavioral assay and data analysis as well as how to prepare the mosquitoes before their introduction into the olfactometer. This uniport olfactometer behavioral assay is currently one of the most reliable methods to study mosquito attraction to a single stimulus.

Odor-evoked transcriptomics of Aedes aegypti mosquitoes.

Modulation of odorant receptors mRNA induced by prolonged odor exposure is highly correlated with ligand-receptor interactions in Drosophila as well as mammals of the Muridae family. If this response feature is conserved in other organisms, this presents a potentially potent initial screening tool when searching for novel receptor-ligand interactions in species with predominantly orphan olfactory receptors. We demonstrate that mRNA modulation in response to 1-octen-3-ol odor exposure occurs in a time- and concentration-dependent manner in Aedes aegypti mosquitoes. To investigate gene expression patterns at a global level, we generated an odor-evoked transcriptome associated with 1-octen-3-ol odor exposure. Transcriptomic data revealed that ORs and OBPs were transcriptionally responsive whereas other chemosensory gene families showed little to no differential expression. Alongside chemosensory gene expression changes, transcriptomic analysis found that prolonged exposure to 1-octen-3-ol modulated xenobiotic response genes, primarily members of the cytochrome P450, insect cuticle proteins, and glucuronosyltransferases families. Together, these findings suggest that mRNA transcriptional modulation caused by prolonged odor exposure is pervasive across taxa and accompanied by the activation of xenobiotic responses. Furthermore, odor-evoked transcriptomics create a potential screening tool for filtering and identification of chemosensory and xenobiotic targets of interest.

Dehydration Dynamics in Terrestrial Arthropods: From Water Sensing to Trophic Interactions.

Since the transition from water to land, maintaining water balance has been a key challenge for terrestrial arthropods. We explore factors that allow terrestrial arthropods to survive within a variably dry world and how they shape ecological interactions. Detection of water and hydration is critical for maintaining water content. Efficient regulation of internal water content is accomplished by excretory and osmoregulatory systems that balance water intake and loss. Biochemical and physiological responses are necessary as water content declines to prevent and repair the damage that occurs during dehydration. Desiccation avoidance can occur seasonally or daily via a move to more favorable areas. Dehydration and its avoidance have ecological impacts that extend beyond a single species to alter trophic interactions. As climate changes, evolutionary and ecological processes will be critical to species survival during drought.

Genetics tools for corpora allata specific gene expression in Aedes aegypti mosquitoes.

Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti.

Carbon dioxide and blood-feeding shift visual cue tracking during navigation in Aedes aegypti mosquitoes.

Haematophagous mosquitoes need a blood meal to complete their reproductive cycle. To accomplish this, female mosquitoes seek vertebrate hosts, land on them and bite. As their eggs mature, they shift attention away from hosts and towards finding sites to lay eggs. We asked whether females were more tuned to visual cues when a host-related signal, carbon dioxide, was present, and further examined the effect of a blood meal, which shifts behaviour to ovipositing. Using a custom, tethered-flight arena that records wing stroke changes while displaying visual cues, we found the presence of carbon dioxide enhances visual attention towards discrete stimuli and improves contrast sensitivity for host-seeking Aedes aegypti mosquitoes. Conversely, intake of a blood meal reverses vertical bar tracking, a stimulus that non-fed females readily follow. This switch in behaviour suggests that having a blood meal modulates visual attention in mosquitoes, a phenomenon that has been described before in olfaction but not in visually driven behaviours.

Skin bacterial volatiles: propelling the future of vector control.

The skin microbiota plays an essential role in the protection against pathogens. It is our skin microbiota that makes us smell different from each other, rendering us more or less attractive to mosquitoes. Mosquitoes exploit skin bacterial odours to locate their hosts and are vectors of pathogens that can cause severe diseases such as malaria and dengue fever. A novel solution for long-lasting protection against insect vectors of disease could be attained by manipulating the bacterial commensals on human skin. The current options for protection against biting insects usually require topical application of repellents that evaporate within hours. We discuss possible routes for the use of commensal bacteria to create a microbial-based repellent.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness.

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

Not Just from Blood: Mosquito Nutrient Acquisition from Nectar Sources.

Anthropophilic female mosquitoes are well known for their strong attraction to human hosts, but plant nectar is a common energy source in their diets. When sugar sources are scarce, female mosquitoes of some species can compensate by taking larger and more frequent blood meals. Male mosquitoes are exclusively dependent on plant nectar or alternative sugar sources. Plant preference is likely driven by an innate attraction that may be enhanced by experience, as mosquitoes learn to recognize available sugar rewards. Nectar-seeking involves the integration of at least three sensory systems: olfaction, vision and taste. The prevention of vector-borne illnesses, the determination of the mosquitoes' ecological role, and the design of efficient sugar-baited traps will all benefit from understanding the molecular basis of nectar-seeking.

Increased temperatures reduce the vectorial capacity of Aedes mosquitoes for Zika virus.

Rapid and significant range expansion of both Zika virus (ZIKV) and its Aedes vector species has resulted in ZIKV being declared a global health threat. Mean temperatures are projected to increase globally, likely resulting in alterations of the transmission potential of mosquito-borne pathogens. To understand the effect of diurnal temperature range on the vectorial capacity of Ae. aegypti and Ae. albopictus for ZIKV, longevity, blood-feeding and vector competence were assessed at two temperature regimes following feeding on infectious blood meals. Higher temperatures resulted in decreased longevity of Ae. aegypti [Log-rank test, χ2, df 35.66, 5, P < 0.001] and a decrease in blood-feeding rates of Ae. albopictus [Fisher's exact test, P < 0.001]. Temperature had a population and species-specific impact on ZIKV infection rates. Overall, Ae. albopictus reared at the lowest temperature regime demonstrated the highest vectorial capacity (0.53) and the highest transmission efficiency (57%). Increased temperature decreased vectorial capacity across groups yet more significant effects were measured with Ae. aegypti relative to Ae. albopictus. The results of this study suggest that future increases in temperature in the Americas could significantly impact vector competence, blood-feeding and longevity, and potentially decrease the overall vectorial capacity of Aedes mosquitoes in the Americas.