Odorant receptors for floral- and plant-derived volatiles in the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae).

Adult mosquitoes require regular sugar meals, usually floral nectar, to survive and flourish in natural habitats. Both males and females locate potential sugar sources using sensory proteins called odorant receptors activated by plant volatiles that facilitate orientation toward flowers or honeydew. The Yellow Fever mosquito, Aedes aegypti (Linnaeus, 1762), possesses a large repertoire of odorant receptors, many of which are likely to support floral odor detection and nectar-seeking. In this study, we have employed a heterologous expression system and the two-electrode voltage clamping technique to identify environmentally relevant chemical compounds that activate specific odorant receptors. Importantly, we have uncovered ligand-receptor pairings for a suite of Aedes aegypti odorant receptors likely to mediate appetitive or aversive behavioral responses, thus shaping a critical aspect of the life history of a medically important mosquito. Moreover, the high degree of conservation of these receptors in other disease-transmitting species suggests common mechanisms of floral odor detection. This knowledge can be used to further investigate mosquito foraging behavior to either enhance existing, or develop novel, control strategies, especially those that incorporate mosquito bait-and-kill or attractive toxic sugar bait technologies.

The evolution of borneol repellency in culicine mosquitoes.

Insects have developed remarkable adaptations to effectively interact with plant secondary metabolites and utilize them as cues to identify suitable hosts. Consequently, humans have used aromatic plants for centuries to repel mosquitoes. The repellent effects of plant volatile compounds are mediated through olfactory structures present in the antennae, and maxillary palps of mosquitoes. Mosquito maxillary palps contain capitate-peg sensilla, which house three olfactory sensory neurons, of which two are mainly tuned to either carbon dioxide or octenol - two animal host odorants. However, the third neuron, which expresses the OR49 receptor, has remained without a known ecologically-relevant odorant since its initial discovery. In this study, we used odorant mixtures and terpenoid-rich Cannabis essential oils to investigate the activation of OR49. Our results demonstrate that two monoterpenoids, borneol and camphor, selectively activate OR49, and OR9-expressing neurons, as well as the MD3 glomerulus in the antennal lobe. We confirm that borneol repels female mosquitoes, and knocking out the gene encoding the OR49 receptor suppresses the response of the corresponding olfactory sensory neuron. Importantly, this molecular mechanism of action is conserved across culicine mosquito species, underscoring its significance in their olfactory systems.

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.

PrimedSherlock: a tool for rapid design of highly specific CRISPR-Cas12 crRNAs.

BACKGROUND: CRISPR-Cas based diagnostic assays provide a portable solution which bridges the benefits of qRT-PCR and serological assays in terms of portability, specificity and ease of use. CRISPR-Cas assays are rapidly fieldable, specific and have been rigorously validated against a number of targets, including HIV and vector-borne pathogens. Recently, CRISPR-Cas12 and CRISPR-Cas13 diagnostic assays have been granted FDA approval for the detection of SARS-CoV-2. A critical step in utilizing this technology requires the design of highly-specific and efficient CRISPR RNAs (crRNAs) and isothermal primers. This process involves intensive manual curation and stringent parameters for design in order to minimize off-target detection while also preserving detection across divergent strains. As such, a single, streamlined bioinformatics platform for rapidly designing crRNAs for use with the CRISPR-Cas12 platform is needed. Here we offer PrimedSherlock, an automated, computer guided process for selecting highly-specific crRNAs and primers for targets of interest. RESULTS: Utilizing PrimedSherlock and publicly available databases, crRNAs were designed against a selection of Flavivirus genomes, including West Nile, Zika and all four serotypes of Dengue. Using outputs from PrimedSherlock in concert with both wildtype A.s Cas12a and Alt-R Cas12a Ultra nucleases, we demonstrated sensitive detection of nucleic acids of each respective arbovirus in in-vitro fluorescence assays. Moreover, primer and crRNA combinations facilitated the detection of their intended targets with minimal off-target background noise. CONCLUSIONS: PrimedSherlock is a novel crRNA design tool, specific for CRISPR-Cas12 diagnostic platforms. It allows for the rapid identification of highly conserved crRNA targets from user-provided primer pairs or PrimedRPA output files. Initial testing of crRNAs against arboviruses of medical importance demonstrated a robust ability to distinguish multiple strains by exploiting polymorphisms within otherwise highly conserved genomic regions. As a freely-accessible software package, PrimedSherlock could significantly increase the efficiency of CRISPR-Cas12 diagnostics. Conceptually, the portability of detection kits could also be enhanced when coupled with isothermal amplification technologies.

Aedes japonicus japonicus in Nebraska and Texas.

Aedes japonicus japonicus continues to spread westward and in this study, its presence is documented in 8 counties in Nebraska and in Bowie County, TX. In 1998, Ae. japonicus was collected in Connecticut, New Jersey, and New York for the 1st records of this species in North America. Except for Louisiana, it has been reported from all states that border or are east of the Mississippi River. In Canada, it has been reported in Ontario and all eastern provinces. In the Pacific Northwest, it has been reported in Washington, Oregon, and British Columbia, and in the midwestern states that do not border the Mississippi River, Kansas, Oklahoma, and South Dakota are the only states to have reported its presence in peer-reviewed journals.

Identification and functional characterization of olfactory indolergic receptors in Musca domestica.

In mosquitoes, indolic compounds are detected by a group of olfactory indolergic Odorant Receptors (indolORs). The ancient origin of indole and 3-methylindole as chemical signals suggest that they may be detected by insects outside the Culicidae clade. To test this hypothesis, we have identified potential indolOR genes in brachyceran flies based on sequence homology. Because of the crucial roles of indolic compounds in oviposition and foraging, we have focused our attention on the housefly Musca domestica. Using a heterologous expression system, we have identified indolOR transcript expression in the female antennae, and have characterized MdomOR30a and MdomOR49b as 3-methylindole and indole receptors, respectively. We have identified a set of 92 putative indolOR genes encoded in the genomes of Culicoidea, Psychodidae and brachycera, described their phylogenetic relationships, and exon/intron structures. Further characterization of indolORs will impact our understanding of insect chemical ecology and will provide targets for the development of novel odor-based tools that can be integrated into existing vector surveillance and control programs.

Ammonium transporter AcAmt mutagenesis uncovers reproductive and physiological defects without impacting olfactory responses to ammonia in the malaria vector mosquito Anopheles coluzzii.

Anopheline mosquitoes are the sole vectors of malaria and rely on olfactory cues for host seeking in which ammonia derived from human sweat plays an essential role. To investigate the function of the Anopheles coluzzii ammonium transporter (AcAmt) in the mosquito olfactory system, we generated an AcAmt null mutant line using CRISPR/Cas9. AcAmt-/- mutants displayed a series of novel phenotypes compared with wild-type mosquitoes including significantly lower insemination rates during mating and increased mortality during eclosion. Furthermore, AcAmt-/- males showed significantly lower sugar consumption while AcAmt-/- females and pupae displayed significantly higher ammonia levels than their wild-type counterparts. Surprisingly, in contrast to previous studies in Drosophila that revealed that the mutation of the ammonium transporter (DmAmt) induces a dramatic reduction of ammonia responses in antennal coeloconic sensilla, no significant differences were observed across a range of peripheral sensory neuron responses to ammonia and other odorants between wild-type and AcAmt-/- females. These data support the existence in mosquitoes of novel compensatory ammonia-sensing mechanisms that are likely to have evolved as a result of the importance of ammonia in host-seeking and other behaviors.

Distribution of Culex coronator in the USA.

In 1920, Culex coronator was reported from San Benito, Texas, and later in Arizona, New Mexico, and Oklahoma. In 2005, this species was reported to be spreading across the southeastern USA. Now reported in 14 states, it has been found as far north as northern Oklahoma; Memphis, TN; and Suffolk, VA. The public health significance of Cx. coronator is not firmly established, even though it has been implicated as a potential vector of several arboviral diseases. This study aims to document additional Cx. coronator county-level records, to provide information about its continued expansion across the southern USA, and to provide a short research update into its vector potential. Data acquired through multistate collaborations and author collections resulted in 146 new county records from Alabama, Arkansas, Florida, Georgia, Louisiana, Mississippi, North Carolina, Oklahoma, South Carolina, and Texas. No new county records were presented for Arizona, New Mexico, Tennessee, or Virginia, which had previously reported this species. With these new data, this species has been documented in 386 counties in 14 states of the continental USA.

Carboxylic acid responses by a conserved odorant receptor in culicine vector mosquitoes.

Many mosquito behaviours that are critical for survival and reproduction depend upon timely responses to chemical cues. Of interest are the effects of volatile organic compounds like carboxylic acids (CAs) that are released by potential blood meal hosts. Short chain CAs are among the primary attractants for host-seeking females and influence host selection in vector species. Although the behavioural relevance of CA's has been established, less is known about the molecular receptive events that evoke responses to specific compounds, with the Ir family of chemoreceptors being broadly implicated in their detection. In this study, we demonstrate that Or orthologs from two vector species, Aedes aegypti (L.) and Aedes albopictus (Skuse), are selectively activated by straight chain carboxylic acids and that these responses are attenuated by the commercial insect repellant N,N-Diethyl-meta-toluamide. Our results suggest that multiple chemoreceptors, representing diverse families, are able to mediate molecular responses to CAs and may therefore underlie important behaviours that directly impact disease-transmission cycles.

Feeding Habits of Vector Mosquitoes in Harris County, TX, 2018.

Mosquito-borne pathogens contribute significantly to the global burden of infectious diseases and are a continuing public health concern in the United States. Blood feeding by vector mosquitoes is a critical step in the transmission of human pathogens. Continuous surveillance of mosquito feeding patterns, especially in major population centers, is necessary for sustainable, effective control strategies. To better understand female feeding habits in Harris County, TX, we trapped mosquitoes from various locations, distributed among urban and semi-urban environments. Bloodmeal hosts were determined using a cytochrome C oxidase I DNA barcoding strategy. We identified a diverse array of vertebrate hosts with a high degree of avian host utilization, most surprisingly from anthropophilic species like Aedes aegypti (L.). We also detected sequences from two different vertebrate hosts in about half of specimens examined, suggesting that multiple bloodmeals had been acquired in the same feeding cycle by a sizable fraction of females in both urban and semi-urban locations. The high proportion of feeding on domestic chickens may indicate that a significant number of homeowners are rearing chickens within close proximity to study trap sites. As non-amplifying hosts, chickens may have a diluting effect on West Nile virus, as well as a zooprophylactic effect in their immediate vicinities. Ultimately, spatial and temporal host utilization patterns add insight into potential disease transmission dynamics, thereby informing vector control strategies in Harris County and other metropolitan areas.

Identification of Mosquito Bloodmeals Collected in Diverse Habitats in Malaysian Borneo Using COI Barcoding.

Land cover and land use change (LCLUC) acts as a catalyst for spillover of arthropod-borne pathogens into novel hosts by shifting host and vector diversity, abundance, and distribution, ultimately reshaping host-vector interactions. Identification of bloodmeals from wild-caught mosquitoes provides insight into host utilization of particular species in particular land cover types, and hence their potential role in pathogen maintenance and spillover. Here, we collected 134 blood-engorged mosquitoes comprising 10 taxa across 9 land cover types in Sarawak, Malaysian Borneo, a region experiencing intense LCLUC and concomitant spillover of arthropod-borne pathogens. Host sources of blood were successfully identified for 116 (87%) mosquitoes using cytochrome oxidase subunit I (COI) barcoding. A diverse range of hosts were identified, including reptiles, amphibians, birds, and mammals. Sixteen engorged Aedes albopictus, a major vector of dengue virus, were collected from seven land cover types and found to feed exclusively on humans (73%) and boar (27%). Culex tritaeniohynchus (n = 2), Cx. gelidus (n = 3), and Cx. quiquefasciatus (n = 3), vectors of Japanese encephalitis virus, fed on humans and pigs in the rural built-up land cover, creating potential transmission networks between these species. Our data support the use of COI barcoding to characterize mosquito-host networks in a biodiversity hotspot.

Heterogeneous expression of the ammonium transporter AgAmt in chemosensory appendages of the malaria vector, Anopheles gambiae.

Ammonia is one of the principal kairomones originating from human and other animal emanations and in that context, plays an essential role in the host-seeking behaviors of the malaria vector mosquito Anopheles gambiae. Nevertheless, despite its importance in directing host-seeking, the mechanisms underlying ammonia detection in the mosquito olfactory system remains largely unknown. In addition to ongoing efforts to identify and characterize the molecular receptors that underlie ammonia sensitivity, previous studies have revealed a prominent role for ammonium transporters (Amt) in modulating antennal and behavioral responses in Drosophila melanogaster and An. gambiae. In the former, localization of DmAmt in antennal sensilla to auxiliary cells surrounding the ammonia sensory neurons led to the hypothesis that its role was to clear excess ammonium ions in the sensillar lymph. In the latter, RT-PCR and heterologous expression have been used to examine the expression and functional characteristics of the An. gambiae ammonium transporter, AgAmt. We now employ advanced transgenic tools to comprehensively examine AgAmt spatial localization across the peripheral chemosensory appendages in larvae and adult female An. gambiae. In the larval antennae, AgAmt appears localized in both neuronal and auxiliary cells. In contrast to D. melanogaster, in the adult antennae, AgAmt-derived signals are observed in both non-neuronal auxiliary cells and in sensory neurons in ammonia-responsive basiconic and coeloconic sensilla. In the maxillary palps, labella, and tarsi, AgAmt appears restricted to sensory neurons. We have also characterized the responses to ammonia of adult antennal coeloconic sensilla and maxillary palp capitate pegs revealing a correlation between sensillar AgAmt expression and ammonia sensitivity. Taken together, these data suggest that AgAmt may play heterogeneous roles in the adult and larval chemosensory apparatus and potentially broad utility as a supra-receptor target in mosquito control.

An odorant receptor from Anopheles gambiae that demonstrates enantioselectivity to the plant volatile, linalool.

Insects express chemical receptors within sensory neurons that are activated by specific cues in the environment, thereby influencing the acquisition of critical resources. A significant gap in our current understanding of insect chemical ecology is defining the molecular mechanisms that underlie sensitivity to plant-emitted volatiles. Linalool is a commonly-occurring monoterpene that has various effects on insect behavior, either acting as an attractant or a repellent, and existing in nature as one of two possible stereoisomers, (R)-(-)-linalool and (S)-(+)-linalool. In this study, we have used a cell-based functional assay to identify linalool and structurally-related compounds as ligands of Odorant receptor 29, a labellum-expressed receptor in the malaria vector mosquito, Anopheles gambiae (AgamOr29). While (R)-(-)-linalool activates AgamOr29, a mixture of the (R) and (S) stereoisomers activates the receptor with higher potency, implying enantiomeric selectivity. Orthologs of Or29 are present in the genomes of Anophelines within the Cellia subgenus. The conservation of this receptor across Anopheline lineages suggests that this ecologically important compound might serve as an attraction cue for nectar-seeking mosquitoes. Moreover, the characterization of a mosquito terpene receptor could serve as a foundation for future ligand-receptor studies of plant volatiles and for the discovery of compounds that can be integrated into push-pull vector control strategies.

Characterization of Chemosensory Responses on the Labellum of the Malaria Vector Mosquito, Anopheles coluzzii.

Anopheles gambiae coluzzii (An. coluzzii) uses olfaction to modulate a range of critical behaviors that are essential for survival and reproduction; most notably, host preference and selection underlie its vectorial capacity for human malaria. As is the case for all mosquitoes, An. coluzzii has three specialized peripheral olfactory appendages-the antennae, maxillary palps and labella-which are used to detect and orient in response to a large variety of olfactory cues. Of these, neither the molecular nor the physiological significance of the labellum have been thoroughly characterized despite suggestions that labial-derived odorant reception is critical for close-range host attraction. Here we report global chemoreceptor transcriptome profiles together with a systematic electrophysiological analysis of labial T2 sensilla, and associated behavioral responses of female An. coluzzii. Single sensillum recordings of the T2 sensilla revealed robust responses to odorants previously associated with human sweat and oviposition sites and identified a 10-component blend that elicited attraction in a dual-choice landing bioassay designed to mimic host seeking in which non-blood fed females were significantly more attracted to the labial-responsive odorant blend as compared to gravid females. Taken together, these data suggest that, in An. coluzzii, olfactory responses derived from the labellum contribute to host-seeking.

Variant Ionotropic Receptors in the Malaria Vector Mosquito Anopheles gambiae Tuned to Amines and Carboxylic Acids.

The principal Afrotropical human malaria vector mosquito, Anopheles gambiae, remains a significant threat to global health. A critical component in the transmission of malaria is the ability of An. gambiae females to detect and respond to human-derived chemical kairomones in their search for blood meal hosts. The basis for host odor responses resides in olfactory receptor neurons (ORNs) that express chemoreceptors encoded by large gene families, including the odorant receptors (ORs) and the variant ionotropic receptors (IRs). While ORs have been the focus of extensive investigation, functional IR complexes and the chemical compounds that activate them have not been identified in An. gambiae. Here we report the transcriptional profiles and functional characterization of three An. gambiae IR (AgIr) complexes that specifically respond to amines or carboxylic acids - two classes of semiochemicals that have been implicated in mediating host-seeking by adult females but are not known to activate An. gambiae ORs (AgOrs). Our results suggest that AgIrs play critical roles in the detection and behavioral responses to important classes of host odors that are underrepresented in the AgOr chemical space.

Disease vectors in the era of next generation sequencing.

Almost 20 % of all infectious human diseases are vector borne and, together, are responsible for over one million deaths per annum. Over the past decade, the decreasing costs of massively parallel sequencing technologies have facilitated the agnostic interrogation of insect vector genomes, giving medical entomologists access to an ever-expanding volume of high-quality genomic and transcriptomic data. In this review, we highlight how genomics resources have provided new insights into the physiology, behavior, and evolution of human disease vectors within the context of the global health landscape.

Antennal-expressed ammonium transporters in the malaria vector mosquito Anopheles gambiae.

The principal Afrotropical malaria vector mosquito, Anopheles gambiae remains a significant threat to human health. In this anthropophagic species, females detect and respond to a range of human-derived volatile kairomones such as ammonia, lactic acid, and other carboxylic acids in their quest for blood meals. While the molecular underpinnings of mosquito olfaction and host seeking are becoming better understood, many questions remain unanswered. In this study, we have identified and characterized two candidate ammonium transporter genes, AgAmt and AgRh50 that are expressed in the mosquito antenna and may contribute to physiological and behavioral responses to ammonia, which is an important host kairomone for vector mosquitoes. AgAmt transcripts are highly enhanced in female antennae while a splice variant of AgRh50 appears to be antennal-specific. Functional expression of AgAmt in Xenopus laevis oocytes facilitates inward currents in response to both ammonium and methylammonium, while AgRh50 is able to partially complement a yeast ammonium transporter mutant strain, validating their conserved roles as ammonium transporters. We present evidence to suggest that both AgAmt and AgRh50 are in vivo ammonium transporters that are important for ammonia sensitivity in An. gambiae antennae, either by clearing ammonia from the sensillar lymph or by facilitating sensory neuron responses to environmental exposure. Accordingly, AgAmt and AgRh50 represent new and potentially important targets for the development of novel vector control strategies.

A blood-free protein meal supporting oogenesis in the Asian tiger mosquito, Aedes albopictus (Skuse).

Female mosquitoes require blood meals to complete oogenesis, or egg development. Current methods of maintaining laboratory colonies of mosquitoes generally rely on the use of whole blood to feed females. Blood feeding protocols require special handling techniques, impart numerous potential health hazards, involve significant costs, and are widely variable in terms of their success rates. In this study, a simple protein formulation was provided to Aedes albopictus using a membrane feeding system. Under the experimental conditions tested, females readily accepted the blood-free meal and produced eggs in greater numbers than cohort females that were fed with whole human blood. Moreover, fertility was comparable between treatments and survivorship of hatched larvae was equal among feedings. This implies that a readily available blood-free meal could be utilized in the laboratory rearing of this species. The elimination of blood handling, reduced cost, and consistency of blood-free meals would potentially be advantageous to mosquito rearing facilities generally, and in terms of scale, to mass rearing facilities specifically.

Odorant receptor-mediated sperm activation in disease vector mosquitoes.

Insects, such as the malaria vector mosquito, Anopheles gambiae, depend upon chemoreceptors to respond to volatiles emitted from a range of environmental sources, most notably blood meal hosts and oviposition sites. A subset of peripheral signaling pathways involved in these insect chemosensory-dependent behaviors requires the activity of heteromeric odorant receptor (OR) ion channel complexes and ligands for numerous A. gambiae ORs (AgOrs) have been identified. Although AgOrs are expressed in nonhead appendages, studies characterizing potential AgOr function in nonolfactory tissues have not been conducted. In the present study, we explore the possibility that AgOrs mediate responses of spermatozoa to endogenous signaling molecules in A. gambiae. In addition to finding AgOr transcript expression in testes, we show that the OR coreceptor, AgOrco, is localized to the flagella of A. gambiae spermatozoa where Orco-specific agonists, antagonists, and other odorant ligands robustly activate flagella beating in an Orco-dependent process. We also demonstrate Orco expression and Orco-mediated activation of spermatozoa in the yellow fever mosquito, Aedes aegypti. Moreover, we find Orco localization in testes across distinct insect taxa and posit that OR-mediated responses in spermatozoa may represent a general characteristic of insect reproduction and an example of convergent evolution.

Review: Improving our knowledge of male mosquito biology in relation to genetic control programmes.

The enormous burden placed on populations worldwide by mosquito-borne diseases, most notably malaria and dengue, is currently being tackled by the use of insecticides sprayed in residences or applied to bednets, and in the case of dengue vectors through reduction of larval breeding sites or larviciding with insecticides thereof. However, these methods are under threat from, amongst other issues, the development of insecticide resistance and the practical difficulty of maintaining long-term community-wide efforts. The sterile insect technique (SIT), whose success hinges on having a good understanding of the biology and behaviour of the male mosquito, is an additional weapon in the limited arsenal against mosquito vectors. The successful production and release of sterile males, which is the mechanism of population suppression by SIT, relies on the release of mass-reared sterile males able to confer sterility in the target population by mating with wild females. A five year Joint FAO/IAEA Coordinated Research Project brought together researchers from around the world to investigate the pre-mating conditions of male mosquitoes (physiology and behaviour, resource acquisition and allocation, and dispersal), the mosquito mating systems and the contribution of molecular or chemical approaches to the understanding of male mosquito mating behaviour. A summary of the existing knowledge and the main novel findings of this group is reviewed here, and further presented in the reviews and research articles that form this Acta Tropica special issue.