Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii.

The attraction of anthropophilic mosquitoes to human host cues, such as body odour and carbon dioxide, gradually increases during adult maturation. This acquisition of host-seeking behaviour correlates with age-dependent changes in odorant receptor (OR) transcript abundance and sensitivity of olfactory sensory neurons (OSNs). One OR gene of the human malaria vector, Anopheles coluzzii, AcolOR39, is significantly downregulated in mature females, and a cognate ligand of AcolOR39, sulcatone, a major component of human emanations, mediates the observed behavioural inhibition of newly emerged (teneral) females to human body odour. Knockout of AcolOR39, using CRISPR-Cas9 mutagenesis, selectively abolished sulcatone detection in OSNs, housed in trichoid sensilla. However, knockout of AcolOR39 altered neither the response rate nor the flight behaviour of teneral females in a wind tunnel, indicating the involvement of other genes, and thus a redundancy, in regulating the acquisition of host seeking in mosquitoes.

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.

A mosquito-specific antennal protein is critical for the attraction to human odor in the malaria vector Anopheles gambiae.

Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.

Non-canonical odor coding in the mosquito.

Aedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

Identification of Bioactive Volatile Organic Compounds Using Combined Gas Chromatography and Single Sensillum Recording.

Most arthropods rely heavily on their sense of smell (i.e., olfaction) to locate and discriminate among mates, food, and egg laying sites. The odors emanating from these resources are composed of blends of volatile compounds that are detected by olfactory sensory neurons (OSNs) that are housed in hair-like structures, called sensilla, on the olfactory organs of arthropods. By inserting an electrode into a single sensillum and recording the activity of the OSNs while stimulating with volatile compounds eluting from a gas chromatograph, combined gas chromatography and single sensillum recording (GC-SSR) provides a high-resolution tool to identify bioactive compounds and to functionally characterize the peripheral olfactory system of arthropods.

Combined Gas Chromatography and Single Sensillum Recording for Mosquitoes.

Combined gas chromatography and single sensillum recording (GC-SSR) joins together the established volatile compound separation and relative quantification techniques of GC with the ability to functionally screen olfactory sensory neurons (OSNs) for volatile selectivity and sensitivity using SSR. With minimal equipment modification, including a splitter and a heated transfer line, half of the effluent from the gas chromatograph column is directed to the flame ionization detector and half to the mosquito antennae. This GC-SSR combination provides a bioassay capable of determining the salient natural ligands for individual OSNs and of providing accurate and comparable dose-response curves irrespective of differences in the volatility of the compounds.

Hyperbolic odorant mixtures as a basis for more efficient signaling between flowering plants and bees.

Animals use odors in many natural contexts, for example, for finding mates or food, or signaling danger. Most analyses of natural odors search for either the most meaningful components of a natural odor mixture, or they use linear metrics to analyze the mixture compositions. However, we have recently shown that the physical space for complex mixtures is 'hyperbolic', meaning that there are certain combinations of variables that have a disproportionately large impact on perception and that these variables have specific interpretations in terms of metabolic processes taking place inside the flower and fruit that produce the odors. Here we show that the statistics of odorants and odorant mixtures produced by inflorescences (Brassica rapa) are also better described with a hyperbolic rather than a linear metric, and that combinations of odorants in the hyperbolic space are better predictors of the nectar and pollen resources sought by bee pollinators than the standard Euclidian combinations. We also show that honey bee and bumble bee antennae can detect most components of the B. rapa odor space that we tested, and the strength of responses correlates with positions of odorants in the hyperbolic space. In sum, a hyperbolic representation can be used to guide investigation of how information is represented at different levels of processing in the CNS.

Reversible plasticity in brain size, behaviour and physiology characterizes caste transitions in a socially flexible ant (Harpegnathos saltator).

Phenotypic plasticity allows organisms to respond to changing environments throughout their lifetime, but these changes are rarely reversible. Exceptions occur in relatively long-lived vertebrate species that exhibit seasonal plasticity in brain size, although similar changes have not been identified in short-lived species, such as insects. Here, we investigate brain plasticity in reproductive workers of the ant Harpegnathos saltator. Unlike most ant species, workers of H. saltator are capable of sexual reproduction, and they compete in a dominance tournament to establish a group of reproductive workers, termed 'gamergates'. We demonstrated that, compared to foragers, gamergates exhibited a 19% reduction in brain volume in addition to significant differences in behaviour, ovarian status, venom production, cuticular hydrocarbon profile, and expression profiles of related genes. In experimentally manipulated gamergates, 6-8 weeks after being reverted back to non-reproductive status their phenotypes shifted to the forager phenotype across all traits we measured, including brain volume, a trait in which changes were previously shown to be irreversible in honeybees and Drosophila. Brain plasticity in H. saltator is therefore more similar to that found in some long-lived vertebrates that display reversible changes in brain volume throughout their lifetimes.

Hold your breath - Differential behavioral and sensory acuity of mosquitoes to acetone and carbon dioxide.

Host seeking in the yellow fever mosquito, Aedes aegypti, and the African malaria mosquito, Anopheles coluzzii, relies on specific and generic host-derived odorants. Previous analyses indicate that the behavioral response of these species depends differentially on the presence of carbon dioxide (CO2) and other constituents in human breath for activation and attraction. In this study, we use a flight tube assay and electrophysiological analysis to assess the role of acetone, a major component of exhaled human breath, in modulating the behavioral and sensory neuronal response of these mosquito species, in the presence and absence of CO2. When presented alone at ecologically relevant concentrations, acetone increases attraction in Ae. aegypti, but not in An. coluzzii. Moreover, in combination with CO2, human breath-equivalents of acetone ranging between 0.1 and 10 ppm reproduces a behavioral response similar to that observed to human breath in host-seeking Ae. aegypti, but not in An. coluzzii. Acetone does, however, reduce attraction to CO2 in An. coluzzii, when presented at a higher concentration of 10 ppm. We identify the capitate peg A neuron of the maxillary palp of both species as a dual detector of CO2 and acetone. The sensory response to acetone, or binary blends of acetone and CO2, reflects the observed behavioral output in both Ae. aegypti and An. coluzzii. We conclude that host recognition is contextual and dependent on a combination of ecologically relevant odorants at naturally occurring concentrations that are encoded, in this case, by differences in the temporal structure of the neuronal response. This information should be considered when designing synthetic blends for that optimally attract mosquitoes for monitoring and control.

Antennal Olfactory Physiology and Behavior of Males of the Ponerine Ant Harpegnathos saltator.

In comparison to the large amount of study on the communication abilities of females in ant societies and their associated chemical ecology and sensory physiology, such study of male ants has been largely ignored; accordingly, little is known about their olfactory sensory capabilities. To address this, we explored peripheral odor sensitivities in male Harpegnathos saltator by measuring the electrophysiological activity of olfactory sensory neurons within antennal trichoid and coeloconic sensilla using an extracellular recording technique. In an initial trial of 46 compounds, sensilla trichodea responded strongly to two alarm pheromone components, while a limited number of non-hydrocarbon odorants elicited strong responses in sensilla coeloconica. Both sensillar types responded indifferently to 31 cuticular hydrocarbons (CHCs) and synthetic long-chain hydrocarbons (HCs) typically found on insect cuticle. In a search for sensilla responding to CHCs and other compounds, we found some sensilla that responded to synthetic HCs and CHCs from virgin queen postpharyngeal glands that are potentially used in close range mate recognition. Olfactometer bioassays of male ants to 15 non-HCs correlated sensory responsiveness to the respective behavioral responses. Comparing olfactory responses between H. saltator males and females, we found that sensilla coeloconica and basiconica of workers showed greater responses and broader selectivity to all compounds. The rarity of CHC-responding trichoid sensilla in Harpegnathos males suggests a more specific role in sexual communication compared to that in females, which use CHCs in a broader communication context.

An Engineered orco Mutation Produces Aberrant Social Behavior and Defective Neural Development in Ants.

Ants exhibit cooperative behaviors and advanced forms of sociality that depend on pheromone-mediated communication. Odorant receptor neurons (ORNs) express specific odorant receptors (ORs) encoded by a dramatically expanded gene family in ants. In most eusocial insects, only the queen can transmit genetic information, restricting genetic studies. In contrast, workers in Harpegnathos saltator ants can be converted into gamergates (pseudoqueens) that can found entire colonies. This feature facilitated CRISPR-Cas9 generation of germline mutations in orco, the gene that encodes the obligate co-receptor of all ORs. orco mutations should significantly impact olfaction. We demonstrate striking functions of Orco in odorant perception, reproductive physiology, and social behavior plasticity. Surprisingly, unlike in other insects, loss of OR functionality also dramatically impairs development of the antennal lobe to which ORNs project. Therefore, the development of genetics in Harpegnathos establishes this ant species as a model organism to study the complexity of eusociality.

Chemosensory sensitivity reflects reproductive status in the ant Harpegnathos saltator.

Insects communicate with pheromones using sensitive antennal sensilla. Although trace amounts of pheromones can be detected by many insects, context-dependent increased costs of high sensitivity might lead to plasticity in sensillum responsiveness. We have functionally characterized basiconic sensilla of the ant Harpegnathos saltator for responses to general odors in comparison to cuticular hydrocarbons which can act as fertility signals emitted by the principal reproductive(s) of a colony to inhibit reproduction by worker colony members. When released from inhibition workers may become reproductive gamergates. We observed plasticity in olfactory sensitivity after transition to reproductive status with significant reductions in electrophysiological responses to several long-chained cuticular hydrocarbons. Although gamergates lived on average five times longer than non-reproductive workers, the shift to reproductive status rather than age differences matched the pattern of changes in olfactory sensitivity. Decreasing sensillum responsiveness to cuticular hydrocarbons could potentially reduce mutually inhibitory or self-inhibitory effects on gamergate reproduction.

Deep-tissue confocal imaging of the central projections of ovipositor sensory afferents in the Egyptian cotton leafworm, Spodoptera littoralis.

The pre-ovipositon behavior of moths is largely dependent upon the cues that a gravid female perceives while assessing potential oviposition sites. Assessment of such sites is accomplished, at least in part, by mechanosensory and gustatory sensilla located on the ovipositor whose sensory neurons project into the terminal abdominal ganglion (TAG). Using anterograde backfill staining, confocal laser scanning microscopy, and three dimensional reconstruction, we traced and analyzed the central projections of the sensory neurons housed in the sensilla located on the ovipositor papillae and explored the neuropilar composition of the TAG in the Egyptian cotton leafworm, Spodoptera littoralis. The TAG consists of three fused neuromeres (6-8th Ner) associated with the 6-8th abdominal segments. Within the TAG, and specifically in the 8th neuromere, four unstructured neuropilar compartments are present; the dorso-ipsilateral motor neuropil (MN), the medio-ipsilateral mechanosensory neuropil (MchN), the medio-ipsilateral small gustatory neuropil (GN), and the medio-contralateral posterior ovipositor glomerulus (Og). The Og appears quite compact, with a hollow core free of terminal arborizations. The MchN is further subdivided into 4 unstructured glomeruli in the 8th neuromere, whose afferents are subsequently extended into 3 glomeruli in the 7th and 6th neuromeres. Few neurites of the Og are populated with large dense varicosities reminiscent of neurosecretory vesicles. Given that all ovipositor nerves converge into a common ganglionic center, the TAG, we assume that this ganglion may be a center for coordination of oviposition behaviors, including movements of the ovipositor during assessment of oviposition substrates and egg laying in S. littoralis.

Physiological organization and topographic mapping of the antennal olfactory sensory neurons in female hawkmoths, Manduca sexta.

The hawkmoth, Manduca sexta, has been a keystone system for developmental, neurobiological, and ecological studies for several decades. Because many of its behaviors are driven by olfactory cues, a thorough understanding of the Manduca olfactory system is essential to studying its biology. With the aim of functionally characterizing single antennal olfactory sensory neurons (OSNs) and determining their detailed topographic location, we performed systematic single-sensillum recordings on 4 morphological types of olfactory sensilla: trichoid-A and -B and basiconic-A and -B. We were able to unambiguously differentiate the colocalized cells associated with single sensilla based on their spike amplitudes. Using a panel of 61 biologically relevant compounds established in behavioral and gas chromatography-electrophysiology experiments, we made 223 recordings from these sensilla. Based on the response spectra of 187 responding OSNs, the sensilla fell into 12 distinct functional classes encompassing 29 OSNs. Selectivity of the 25 responding OSNs varied from narrowly tuned (responding to only one or a subset of compounds), to very broadly tuned (responding to multiple compounds), in a concentration-dependent manner. Four OSNs, however, did not respond to the tested components. Topographic mapping of the sensilla revealed that some physiological sensillum types are confined to particular locations on the antennal surface while other classes are more or less irregularly scattered all over the antennal annuli. Such information will prove beneficial for future receptor deorphanization, in situ hybridization, and molecular manipulation experiments.

Natural odor ligands for olfactory receptor neurons of the female mosquito Aedes aegypti: use of gas chromatography-linked single sensillum recordings.

Female Aedes aegypti are vectors of dengue and yellow fever. Odor volatiles are the predominant cues that drive the host-seeking behavior of Ae. aegypti. Odorant molecules are detected and discriminated by olfactory receptor neurons (ORNs) housed in sensory hairs, sensilla, located on the antennae and maxillary palps. In a previous study, we used odor volatiles that are behaviorally and/or electrophysiologically active for Ae. aegypti and other mosquito species to show that antennal ORNs of female Ae. aegypti are divided into functionally different classes. In the present study, we have, for the first time, conducted gas chromatography-coupled single sensillum recordings (GC-SSR) from antennal trichoid and intermediate sensilla of female Ae. aegypti in order to screen for additional putative host attractants and repellents. We used headspace collections from biologically relevant sources, such as different human body parts (including feet, trunk regions and armpit), as well as a plant species used as a mosquito repellent, Nepeta faassenii. We found that a number of ORN types strongly responded to one or more of the biological extracts. GC-SSR recordings revealed several active components, which were subsequently identified through GC-linked mass spectrometry (GC-MS). Electrophysiologically active volatiles from human skin included heptanal, octanal, nonanal and decanal.

The antennal lobe of the African malaria mosquito, Anopheles gambiae - innervation and three-dimensional reconstruction.

Antibody labelling and subsequent three-dimensional reconstructions of the primary olfactory centres, the antennal lobes, of male and female African malaria mosquitoes, Anopheles gambiae, revealed 61 and 60 glomerular neuropils respectively. In addition to the small difference in number of glomeruli, sexual dimorphism was observed in both the size of the antennal lobe and of individual glomeruli. Furthermore, sexual specificity was observed within the array. Anterograde staining of afferents from peripheral olfactory organs support the reconstruction of the glomerular array. Although anterograde stainings support an organotopic organization of the antennal lobe, convergence of afferents originating from different organs into single glomeruli is observed. This finding, in both A. gambiae and A. aegypti, may shed new light upon the development and function of the olfactory system.

Functional classification and central nervous projections of olfactory receptor neurons housed in antennal trichoid sensilla of female yellow fever mosquitoes, Aedes aegypti.

Mosquitoes are highly dependent on their olfactory system for, e.g. host location and identification of nectar-feeding and oviposition sites. Odours are detected by olfactory receptor neurons (ORNs) housed in hair-shaped structures, sensilla, on the antennae and maxillary palps. In order to unravel the function of the olfactory system in the yellow fever vector, Aedes aegypti, we performed single-sensillum recordings from trichoid sensilla on female antennae. These sensilla are divided into four distinct morphological types. Based on the response to a set of 16 odour compounds, we identified 18 different ORN types, housed in 10 sensillum types. The ORNs responded to behaviourally relevant olfactory cues, such as oviposition attractants and sweat-borne compounds, including 4-methylcyclohexanol and indole, respectively. Two ORNs housed in these sensilla, as well as two ORNs housed in an additional sensillum type, did not respond to any of the compounds tested. The ORNs housed in individual sensilla exhibited stereotypical pairing and displayed differences in signalling mode (excitatory and inhibitory) as well as in temporal response patterns. In addition to physiological characterization, we performed anterograde neurobiotin stainings of functionally identified ORNs in order to define the functional map among olfactory glomeruli in the primary olfactory centre, the antennal lobe. The targeted glomeruli were compared with an established 3D map. Our data showed that the ORN types sent their axons to defined antennal lobe glomeruli in a stereotypic pattern.

Neuronal architecture of the mosquito deutocerebrum.

Mosquito behavior is heavily dependent on olfactory and mechanosensory cues, which are detected by receptor neurons on the antenna and on the palps. Recent progress in mosquito sensory genomics highlights the need for an up-to-date understanding of the neural architecture of the mosquito brain. Here we present a detailed description of the neural structure of the primary target of the majority of these neurons, the deutocerebrum, in the African malaria (Anopheles gambiae) and yellow fever (Aedes aegypti) mosquitoes. Special focus is made on the olfactory system, the antennal lobe (AL), where we present high-resolution three-dimensional models of the ALs of male and female Ae. aegypti. These models reveal a sexual dimorphism in the number of glomeruli, 49 and 50 glomeruli in male and female mosquitoes, respectively, and in the size of several of the identified glomeruli. The fine structure of receptor neuron terminations in the AL and the rest of the deutocerebrum is described, as are the arborizations of intrinsic deutocerebral neurons and neurons providing output to higher brain areas. In the AL a specific and very large center receiving input from the mechanosensory Johnston's organ is revealed as a multilobed structure receiving peripheral input according to a somatotopic pattern. Within the antennal nerve a specific neuropil containing early, bouton-like ramifications of receptor neurons is described. Within the glomerular array of the AL, neurons providing a possible feedback circuit to antennal receptor neurons are shown. With these results we provide a new resolution in mosquito deutocerebral architecture.