Stop the biting: targeting a mosquito's sense of smell.

Mosquitoes are a great threat to human health. Fortunately, they have a weakness: they utilize their sense of smell to target a human host. Recent studies examine the effectiveness of protecting humans from attack by ablating or odorant targeting mosquito olfactory receptors. The results are both promising and alarming.

The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.

We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This "Q system" offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional "logic gates" with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis.

Olfaction in Anopheles mosquitoes.

As vectors of disease, mosquitoes are a global threat to human health. The Anopheles mosquito is the deadliest mosquito species as the insect vector of the malaria-causing parasite, which kills hundreds of thousands every year. These mosquitoes are reliant on their sense of smell (olfaction) to guide most of their behaviors, and a better understanding of Anopheles olfaction identifies opportunities for reducing the spread of malaria. This review takes a detailed look at Anopheles olfaction. We explore a range of topics from chemosensory receptors, olfactory neurons, and sensory appendages to behaviors guided by olfaction (including host-seeking, foraging, oviposition, and mating), to vector management strategies that target mosquito olfaction. We identify many research areas that remain to be addressed.

Insect repellents mediate species-specific olfactory behaviours in mosquitoes.

BACKGROUND: The species-specific mode of action for DEET and many other mosquito repellents is often unclear. Confusion may arise for many reasons. First, the response of a single mosquito species is often used to represent all mosquito species. Second, behavioural studies usually test the effect of repellents on mosquito attraction towards human odorants, rather than their direct repulsive effect on mosquitoes. Third, the mosquito sensory neuron responses towards repellents are often not directly examined. METHODS: A close proximity response assay was used to test the direct repulsive effect of six mosquito repellents on Anopheles coluzzii, Aedes aegypti and Culex quinquefasciatus mosquitoes. Additionally, the behavioural assay and calcium imaging recordings of antennae were used to test the response of An. coluzzii mosquitoes towards two human odorants (1-octen-3-ol and benzaldehyde) at different concentrations, and mixtures of the repellents lemongrass oil and p-menthane-3,8-diol (PMD) with DEET. RESULTS: Anopheles coluzzii mosquitoes were repelled by lemongrass oil and PMD, while Ae. aegypti and Cx. quinquefasciatus mosquitoes were repelled by lemongrass oil, PMD, eugenol, and DEET. In addition, high concentrations of 1-octen-3-ol and benzaldehyde were repellent, and activated more olfactory receptor neurons on the An. coluzzii antennae than lower concentrations. Finally, changes in olfactory responses to repellent mixtures reflected changes in repulsive behaviours. CONCLUSIONS: The findings described here suggest that different species of mosquitoes have different behavioural responses to repellents. The data further suggest that high-odour concentrations may recruit repellent-sensing neurons, or generally excite many olfactory neurons, yielding repellent behavioural responses. Finally, DEET can decrease the neuronal and behavioural response of An. coluzzii mosquitoes towards PMD but not towards lemongrass oil. Overall, these studies can help inform mosquito repellent choice by species, guide decisions on effective repellent blends, and could ultimately identify the olfactory neurons and receptors in mosquitoes that mediate repellency.

Improved and expanded Q-system reagents for genetic manipulations.

The Q system is a repressible binary expression system for transgenic manipulations in living organisms. Through protein engineering and in vivo functional tests, we report here variants of the Q-system transcriptional activator, including QF2, for driving strong and ubiquitous expression in all Drosophila tissues. Our QF2, Gal4QF and LexAQF chimeric transcriptional activators substantially enrich the toolkit available for transgenic regulation in Drosophila melanogaster.

Farnesol-detecting olfactory neurons in Drosophila.

We set out to deorphanize a subset of putative Drosophila odorant receptors expressed in trichoid sensilla using a transgenic in vivo misexpression approach. We identified farnesol as a potent and specific activator for the orphan odorant receptor Or83c. Farnesol is an intermediate in juvenile hormone biosynthesis, but is also produced by ripe citrus fruit peels. Here, we show that farnesol stimulates robust activation of Or83c-expressing olfactory neurons, even at high dilutions. The CD36 homolog Snmp1 is required for normal farnesol response kinetics. The neurons expressing Or83c are found in a subset of poorly characterized intermediate sensilla. We show that these neurons mediate attraction behavior to low concentrations of farnesol and that Or83c receptor mutants are defective for this behavior. Or83c neurons innervate the DC3 glomerulus in the antennal lobe and projection neurons relaying information from this glomerulus to higher brain centers target a region of the lateral horn previously implicated in pheromone perception. Our findings identify a sensitive, narrowly tuned receptor that mediates attraction behavior to farnesol and demonstrates an effective approach to deorphanizing odorant receptors expressed in neurons located in intermediate and trichoid sensilla that may not function in the classical "empty basiconic neuron" system.

Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans.

We established a transcription-based binary gene expression system in Caenorhabditis elegans using the recently developed Q system. This system, derived from genes in Neurospora crassa, uses the transcriptional activator QF to induce the expression of target genes. Activation can be efficiently suppressed by the transcriptional repressor QS, and suppression can be relieved by the nontoxic small molecule quinic acid. We used QF, QS and quinic acid to achieve temporal and spatial control of transgene expression in various tissues in C. elegans. We also developed a split Q system, in which we separated QF into two parts encoding its DNA-binding and transcription-activation domains. Each domain showed negligible transcriptional activity when expressed alone, but expression of both reconstituted QF activity, providing additional combinatorial power to control gene expression.

Adoption of the Q transcriptional regulatory system for zebrafish transgenesis.

The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio rerio. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos; however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish.

A versatile in vivo system for directed dissection of gene expression patterns.

Tissue-specific gene expression using the upstream activating sequence (UAS)­GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.

Hybridization Chain Reaction RNA Whole-Mount Fluorescence In situ Hybridization of Chemosensory Genes in Mosquito Olfactory Appendages.

Mosquitoes are effective vectors of deadly diseases and can navigate their chemical environment using chemosensory receptors expressed in their olfactory appendages. Understanding how chemosensory receptors are spatially organized in the peripheral olfactory appendages can offer insights into how odor is encoded in the mosquito olfactory system and inform new ways to combat the spread of mosquito-borne diseases. The emergence of third-generation hybridization chain reaction RNA whole-mount fluorescence in situ hybridization (HCR RNA WM-FISH) allows for spatial mapping and simultaneous expression profiling of multiple chemosensory genes. Here, we describe a stepwise approach for performing HCR RNA WM-FISH on the Anopheles mosquito antenna and maxillary palp. We investigated the sensitivity of this technique by examining the expression profile of ionotropic olfactory receptors. We asked if the HCR WM-FISH technique described was suitable for multiplexed studies by tethering RNA probes to three spectrally distinct fluorophores. Results provided evidence that HCR RNA WM-FISH is robustly sensitive to simultaneously detect multiple chemosensory genes in the antenna and maxillary palp olfactory appendages. Further investigations attest to the suitability of HCR WM-FISH for co-expression profiling of double and triple RNA targets. This technique, when applied with modifications, could be adaptable to localize genes of interest in the olfactory tissues of other insect species or in other appendages.

A spatial map of antennal-expressed ionotropic receptors in the malaria mosquito.

The mosquito's antenna represents its main olfactory appendage for detecting volatile chemical cues from the environment. Whole-mount fluorescence in situ hybridization of ionotropic receptors (IRs) expressed in the antennae reveals that the antenna might be divisible into proximal and distal functional domains. The number of IR-positive cells appear stereotyped within each antennal segment (flagellomere). Highly expressed odor-tuning IRs exhibit distinct co-localization patterns with the IR coreceptors Ir8a, Ir25a, and Ir76b that might predict their functional properties. Genetic knockin and in vivo functional imaging of IR41c-expressing neurons indicate both odor-induced activation and inhibition in response to select amine compounds. Targeted mutagenesis of IR41c does not abolish behavioral responses to the amine compounds. Our study provides a comprehensive map of IR-expressing neurons in the main olfactory appendage of mosquitoes. These findings show organizing principles of Anopheles IR-expressing neurons, which might underlie their functional contribution to the detection of behaviorally relevant odors.

Neurogenetic identification of mosquito sensory neurons.

Anopheles mosquitoes, as vectors for the malaria parasite, are a global threat to human health. To find and bite a human, they utilize neurons within their sensory appendages. However, the identity and quantification of sensory appendage neurons are lacking. Here we use a neurogenetic approach to label all neurons in Anopheles coluzzii mosquitoes. We utilize the homology assisted CRISPR knock-in (HACK) approach to generate a T2A-QF2w knock-in of the synaptic gene bruchpilot. We use a membrane-targeted GFP reporter to visualize the neurons in the brain and to quantify neurons in all major chemosensory appendages (antenna, maxillary palp, labella, tarsi, and ovipositor). By comparing labeling of brp>GFP and Orco>GFP mosquitoes, we predict the extent of neurons expressing ionotropic receptors (IRs) or other chemosensory receptors. This work introduces a valuable genetic tool for the functional analysis of Anopheles mosquito neurobiology and initiates characterization of the sensory neurons that guide mosquito behavior.

Calcium Imaging of Anopheles coluzzii Mosquito Antennae Expressing the Calcium Indicator GCaMP6f.

Calcium imaging is a technique used to measure functional neuronal activities in response to stimuli. It has been used for years to study odorant-induced responses in insects (i.e., honeybees, Drosophila, and moths) and was recently introduced into mosquitoes. Traditionally, calcium imaging in mosquitoes was performed using nonspecific calcium indicator dyes to examine neuronal responses in whole insect brain regions, but the development of genetically encoded calcium indicators (GECIs) has facilitated the ability to perform functional calcium imaging on specific tissues. For example, by specifically expressing a GECI in olfactory neurons, the odor-induced responses of these neurons in peripheral organs can be examined. Calcium imaging of mosquito antennae further provides an advantageous method for simultaneously visualizing the activity of several antennal neurons in a single experiment. In this protocol, we describe a calcium imaging method to study odor-evoked responses in Anopheles coluzzii antennae expressing the calcium indicator GCaMP6f. This method requires imaging equipment (compound microscope, light sources, and camera), an odorant delivery system, and image acquisition software. The mosquito preparation is straightforward but requires practice to minimize mosquito movement during imaging. Recorded videos can be analyzed using Fiji software to generate heatmaps and activity traces for odorant-evoked responses. This protocol can also be used, with some modifications, to study other peripheral organs (such as labella, palps, and tarsi).

Genetically Encoded Calcium Indicators for Functional Imaging of Mosquito Olfactory Neurons.

Mosquitoes transmit a multitude of diseases to humans and animals through biting and blood feeding. To locate their hosts, mosquitoes primarily use their sense of smell. Therefore, an understanding of mosquito olfaction will help develop strategies to control the diseases they transmit. A mosquito's sense of smell is determined by the response of olfactory neurons on its peripheral olfactory organs. Traditionally, mosquito olfactory neuron activity has been examined using electrophysiological techniques such as electroantennography and single sensillum recordings. Electroantennography examines if an odorant is detectable by the ensemble of all antennal neurons. In contrast, single sensillum electrophysiology allows detailed recordings of the activity of two to three neurons at a time. However, single sensillum recording of olfactory neurons is difficult, laborious, and typically allows examination of only a few neurons on the antenna. A promising new approach is to use optical imaging techniques to provide a way to visualize the global response of olfactory organs to an odor, as well as the specific responses of several olfactory neurons to that odor. In particular, calcium imaging has progressed significantly, from the use of chemical calcium indicators to the development of genetically encoded calcium sensors. These advances have opened the way to study the mode of action of known mosquito attractants and repellents as well as a way to screen potential new attractants and repellents. Here, we provide an introduction to the different types of calcium indicators and their uses for investigating the function of mosquito sensory neurons.

Chemosensory ionotropic receptors in human host-seeking mosquitoes.

Half the world's human population is at risk for mosquito-borne diseases. Mosquitoes rely mainly on their sense of smell to find a vertebrate blood host, nectar source, and a suitable oviposition site. Advances in neurogenetic tools have now aided our understanding of the receptors that mediate the detection of sensory cues that emanate from humans. Recent studies in the anthropophilic mosquito vectors, Aedes aegypti and Anopheles gambiae, have implicated the chemosensory ionotropic-receptor (IR) family in the detection of behaviorally relevant odors and uncovered functions beyond chemical sensing. Here, we highlight the multifunctional roles of the chemosensory ionotropic receptors in anthropophilic mosquito vectors and suggest future directions to improve our understanding of the IR family.

Odorant-receptor-mediated regulation of chemosensory gene expression in the malaria mosquito Anopheles gambiae.

Mosquitoes locate and approach humans based on the activity of odorant receptors (ORs) expressed on olfactory receptor neurons (ORNs). Olfactogenetic experiments in Anopheles gambiae mosquitoes revealed that the ectopic expression of an AgOR (AgOR2) in ORNs dampened the activity of the expressing neuron. This contrasts with studies in Drosophila melanogaster in which the ectopic expression of non-native ORs in ORNs confers ectopic neuronal responses without interfering with native olfactory physiology. RNA-seq analyses comparing wild-type antennae to those ectopically expressing AgOR2 in ORNs indicated that nearly all AgOR transcripts were significantly downregulated (except for AgOR2). Additional experiments suggest that AgOR2 protein rather than mRNA mediates this downregulation. Using in situ hybridization, we find that AgOR gene choice is active into adulthood and that AgOR2 expression inhibits AgORs from turning on at this late stage. Our study shows that the ORNs of Anopheles mosquitoes (in contrast to Drosophila) are sensitive to a currently unexplored mechanism of AgOR regulation.

The Q-system: A Versatile Repressible Binary Expression System.

Binary expression systems are useful genetic tools for experimentally labeling or manipulating the function of defined cells. The Q-system is a repressible binary expression system that consists of a transcription factor QF (and the recently improved QF2/QF2w), the inhibitor QS, a QUAS-geneX effector, and a drug that inhibits QS (quinic acid). The Q-system can be used alone or in combination with other binary expression systems, such as GAL4/UAS and LexA/LexAop. In this review chapter, we discuss the past, present, and future of the Q-system for applications in Drosophila and other organisms. We discuss the in vivo application of the Q-system for transgenic labeling, the modular nature of QF that allows chimeric or split transcriptional activators to be developed, its temporal control by quinic acid, new methods to generate QF2 reagents, intersectional expression labeling, and its recent adoption into many emerging experimental species.

Chemoreceptor co-expression in Drosophila melanogaster olfactory neurons.

Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila, the odorant receptors (ORs) and the ionotropic receptors (IRs). The dozens of odorant-binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a, Ir8a, and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in D. melanogaster (Orco, Ir8a, Ir76b, Ir25a). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco, Ir8a, and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of Orco and Ir25a in Drosophila sechellia and Anopheles coluzzii olfactory neurons. These results suggest that co-expression of chemosensory receptors is common in insect olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.

MicroRNA processing pathway regulates olfactory neuron morphogenesis.

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs [1, 2]. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes [3, 4]. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition (Figure 1A). Here, we identify two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections [5, 6]. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers [7-9]. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. Our findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system.

Akt regulates growth by directly phosphorylating Tsc2.

The direct mechanism by which the serine/threonine kinase Akt (also known as protein kinase B (PKB)) regulates cell growth is unknown. Here, we report that Drosophila melanogaster Akt/PKB stimulates growth by phosphorylating the tuberous sclerosis complex 2 (Tsc2) tumour suppressor and inhibiting formation of a Tsc1-Tsc2 complex. We show that Akt/PKB directly phosphorylates Drosophila Tsc2 in vitro at the conserved residues, Ser 924 and Thr 1518. Mutation of these sites renders Tsc2 insensitive to Akt/PKB signalling, increasing the stability of the Tsc1-Tsc2 complex within the cell. Stimulating Akt/PKB signalling in vivo markedly increases cell growth/size, disrupts the Tsc1-Tsc2 complex and disturbs the distinct subcellular localization of Tsc1 and Tsc2. Furthermore, all Akt/PKB growth signals are blocked by expression of a Tsc2 mutant lacking Akt phosphorylation sites. Thus, Tsc2 seems to be the critical target of Akt in mediating growth signals for the insulin signalling pathway.