Mosquito Antennae Research Articles

Repellency, toxicity, and physiological actions of low molecular weight basic amines in mosquitoes.

This study investigated the behavioral responses and toxicity of three basic amines: 1-methylpiperazine, 1-methylpyrrolidine, and triethylamine (TEA), compounds suggested previously to be anosmic in vapor exposures to caged mosquitoes. These compounds showed repellency of Aedes aegypti mosquitoes, followed by flightlessness, knockdown, and paralysis, all increasing with exposure time and dosage. Electrophysiological experiments showed a blocking effect on nerve discharge of the Drosophila melanogaster larval central nervous system (CNS) with little evidence of hyperexcitation. Blockage of voltage-gated (Kv2) potassium channel currents under patch clamp occurred at similar concentrations. Involvement of K+ channels in the action of basic amines was supported by behavior and CNS recordings of a Shaker Kv1 mutant exposed to TEA, where instead of blockage, a hyperexcitation of nerve firing was observed. Experiments on cockroach leg mechanoreceptors demonstrated neuronal excitation and on mosquito antennae strong electroantennogram (EAG) signals with an augmentation of blank air responses after a single puff of basic amine. The neurophysiological effects of basic amines are consistent with K+ channel block, whereas the antennal EAG response was not obviously associated with anosmia. The low-dose effects of basic amines appear to be repellency and bradykinesia. Overall, the findings provide key insights into the mechanisms underlying the biological activity of basic amines. © 2024 Society of Chemical Industry.

Identifikasi Tumbuhan sebagai Repellent terhadap Nyamuk : Review

Indonesia as one of the tropical countries in the world with high air humidity triggers the breeding of mosquitoes such as Aedes aegypti which is one of the vectors of Dengue Hemorrhagic Fever (DHF). This type of research is a literature study using the literature review method. This method is carried out by evaluating, interpreting and explaining the ideas discussed in several studies by searching for sources from the Google Scholar database. The selection of the type of article is based on the year of publication, the article can be accessed in its entirety, and discusses plants as repellents against mosquitoes. Based on research results from several sources, there is a biological prospect as an anti-mosquito agent for anti-mosquito and repellent/repelling power because it contains bioactive compounds, such as alkaloids, saponins, flavonoids, phenols, tannins, steroids and terpenoids. These compounds enter through respiratory contact and digestive/gastric toxins so that mosquitoes experience mortality/death. This compound also blocks chemical receptors (chemoreceptors) found on the palpus and antennae of mosquitoes, so that mosquitoes escape without sucking blood again. It is hoped that this review article can provide information regarding repellent plants as anti-mosquitoes. It can be concluded from the results of the review that there are plants that have repellent power against Aedes aegypti with increased protective power according to the increase in concentration of the plant used, as well as the length of exposure of the extract from each plant to the skin.

Morphological characterization of antennal sensilla in Toxorhynchites theobaldi, Toxorhynchites violaceus, andLutziabigotiadults: a comparative study using scanning electron microscopy.

Some mosquitoes, including species of the genus Toxorhynchites, are known for actively preying on other mosquito larvae, making these predators valuable allies in the fight against vector-borne diseases. A comprehensive understanding of the anatomy and physiology of these potential biological control agents is helpful for the development of effective strategies for controlling vector populations. This includes the antennae, a crucial component in the search for hosts, mating, and selection of oviposition sites. This study utilized scanning electron microscopy to characterize the sensilla on the antennae of adult mosquitoes from two species that are exclusively phytophagous, including Toxorhynchites theobaldi and Toxorhynchites violaceus, as well asLutzia bigoti, which females are allegedly hematophagous. The types of sensilla in each species were compared, and five basic types of antennal sensilla were identified: trichoid, chaetic, coeloconic, basiconic, and ampullacea. The analysis also found that they were morphologically similar across the three species, regardless of feeding habits or sex. The identification and characterization of basic types of antennal sensilla in T. theobaldi, T. violaceus, andL. bigoti suggest that these structures, which play a crucial role in the behavior and ecology, have common functions across different mosquito species, despite differences in feeding habits or sex.

Tiny spies: mosquito antennae are sensitive sensors for eavesdropping on frog calls.

Most mosquito and midge species use hearing during acoustic mating behaviors. For frog-biting species, however, hearing plays an important role beyond mating as females rely on anuran calls to obtain blood meals. Despite the extensive work examining hearing in mosquito species that use sound in mating contexts, our understanding of how mosquitoes hear frog calls is limited. Here, we directly investigated the mechanisms underlying detection of frog calls by a mosquito species specialized on eavesdropping on anuran mating signals: Uranotaenia lowii. Behavioral, biomechanical and neurophysiological analyses revealed that the antenna of this frog-biting species can detect frog calls by relying on neural and mechanical responses comparable to those of non-frog-biting species. Our findings show that in Ur. lowii, contrary to most species, males do not use sound for mating, but females use hearing to locate their anuran host. We also show that the response of the antennae of this frog-biting species resembles that of the antenna of species that use hearing for mating. Finally, we discuss our data considering how mosquitoes may have evolved the ability to tap into the communication system of frogs.

Mapping the Auditory Space of Culex pipiens Female Mosquitoes in 3D.

The task of directional hearing faces most animals that possess ears. They approach this task in different ways, but a common trait is the use of binaural cues to find the direction to the source of sound. In insects, the task is further complicated by their small size and, hence, minute temporal and level differences between two ears. A single symmetric flagellar particle velocity receiver, such as the antenna of a mosquito, should not be able to discriminate between the two opposite directions along the vector of the sound wave. Paired antennae of mosquitoes presume the usage of binaural hearing, but its mechanisms are expected to be significantly different from the ones typical for the pressure receivers. However, the directionality of flagellar auditory organs has received little attention. Here, we measured the in-flight orientation of antennae in female Culex pipiens pipiens mosquitoes and obtained a detailed physiological mapping of the Johnston's organ directionality at the level of individual sensory units. By combining these data, we created a three-dimensional model of the mosquito's auditory space. The orientation of the antennae was found to be coordinated with the neuronal asymmetry of the Johnston's organs to maintain a uniformly shaped auditory space, symmetric relative to a flying mosquito. The overlap of the directional characteristics of the left and right sensory units was found to be optimal for binaural hearing focused primarily in front of, above and below a flying mosquito.

A highly expressed odorant receptor from the yellow fever mosquito, AaegOR11, responds to (+)- and (−)-fenchone and a phenolic repellent

The cornerstone of the reverse chemical ecology approach is the identification of odorant receptors (OR) sensitive to compounds in a large panel of odorants. In this approach, we de-orphanize ORs and, subsequently, measure behaviors elicited by these semiochemicals. After that, we evaluate behaviorally active compounds for applications in insect vector management. Intriguingly, multiple ORs encoded by genes highly expressed in mosquito antennae do not respond to any test odorant. One such case is CquiOR125 from the southern house mosquito, Culex quinquefasciatus Say. To better understand CquiOR125's role in Culex mosquito olfaction, we have cloned a CquiOR125 orthologue in the genome of the yellow fever mosquito, Aedes aegypti (L.), AaegOR11. Unlike the unresponsive nature of the orthologue in Cx. quinquefasciatus, oocytes co-expressing AaegOR11 and AaegOrco elicited robust responses when challenged with fenchone, 2,3-dimethylphenol, 3,4-dimethylphenol, 4-methycyclohexanol, and acetophenone. Interestingly, AaegOR11 responded strongly and equally to (+)- and (−)-fenchone, with no chiral discrimination. Contrary to reports in the literature, fenchone did not show any repellency activity against Ae. aegypti or Cx. quinquefasciatus. Laboratory and field tests did not show significant increases in egg captures in cups filled with fenchone solutions compared to control cups. The second most potent ligand, 2,3-dimethylphenol, showed repellency activity stronger than that elicited by DEET at the same dose. We, therefore, concluded that AaegOR11 is a mosquito repellent sensor. It is feasible that CquiOR125 responds to repellents that remain elusive.

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).

RNA In Situ Hybridization of Whole-Mount Mosquito Olfactory Tissues.

This protocol presents a method for RNA in situ hybridization of whole-mounted preparations of mosquito antennae and maxillary palps. It can be adapted for other peripheral appendages such as the legs or proboscis.

Immunostaining Whole-Mount Olfactory Tissues of Mosquitoes.

Mosquito olfactory tissues are covered in fine hair-like sensory structures called sensilla that house olfactory sensory neurons. This protocol for immunostaining whole-mounted mosquito antennae and maxillary palps enables the visualization of these neurons.

Mosquito Electroantennogram Recordings.

Electroantennography (EAG) is a simple, yet powerful technique that can be used to indicate that chemical compounds are detected by mosquito antennae. When coupled with gas chromatography (GC), it allows for the identification of detectable compounds within complex mixtures, including those in plant and animal headspaces that might influence mosquito behavior. EAGs and GC-EAGs reveal compounds that can be tested in behavioral assays with the goal of developing new repellents or attractants for mosquito control.

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.

The Peptide Hormone CNMa Influences Egg Production in the Mosquito Aedes aegypti.

Simple SummaryHormones are important signaling molecules mediating insect reproduction that require specific receptors to transmit their signal across the plasma membrane of target cells. While the receptors many hormones bind to are known, some receptors are “orphans” with unknown ligands. The hormone CNMa was previously found to bind a specific receptor in fruit flies. Two copies of the receptor are found in mosquitoes, but only one gene for the hormone is present. We found that both receptors in the yellow fever mosquito are activated by CNMa. One of the receptors is expressed in mosquito ovaries, which to us implies that the hormone and its receptors may be involved in mosquito reproduction. We further found that injecting the peptide into females reduced the number of eggs that were laid. These experiments suggest that CNMa may be an important factor governing mosquito reproduction.Mosquito reproduction is regulated by a suite of hormones, many acting through membrane-bound receptor proteins. The Aedes aegypti G protein-coupled receptors AAEL024199 (AeCNMaR-1a) and AAEL018316 (AeCNMaR-1b) were identified as orthologs of the Drosophila melanogaster CNMa receptor (DmCNMaR). The receptor was duplicated early in the evolution of insects, and subsequently in Culicidae, into what we refer to as CNMaR-1a and CNMaR-1b. AeCNMaR-1a is only detected in male mosquito antennae while AeCNMaR-1b is expressed at high levels in mosquito ovaries. Using a heterologous cell assay, we determined that AeCNMa activates AeCNMaR-1a with a ~10-fold lower concentration than it does AeCNMaR-1b, though both receptors displayed half maximal effective concentrations of AeCNMa in the low nanomolar range. Finally, we show that injections of AeCNMa into blood-fed mated female Ae. aegypti resulted in fewer eggs laid.

Bioallethrin activates specific olfactory sensory neurons and elicits spatial repellency in Aedes aegypti.

Use of pyrethroid insecticides is a pivotal strategy for mosquito control globally. Commonly known for their insecticidal activity by acting on voltage-gated sodium channels, pyrethroids, such as bioallethrin and transfluthrin, are used in mosquito coils, emanators and other vaporizers to repel mosquitoes and other biting arthropods. However, whether specific olfactory receptor neurons are activated by pyrethroids to trigger spatial repellency remains unknown. We used behavioral and electrophysiological approaches to elucidate the mechanism of bioallethrin repellency in Aedes aegypti, a major vector of dengue, yellow fever, Zika and chikungunya viruses. We found that bioallethrin elicits spatial (i.e. non-contact) repellency and activates a specific type of olfactory receptor neuron in mosquito antennae. Furthermore, bioallethrin repellency is significantly reduced in a mosquito mutant of Orco, an obligate olfactory co-receptor that is essential for the function of odorant receptors (Ors). These results indicate that activation of specific Or(s) by bioallethrin contributes to bioallethrin repellency. In addition, bioallethrin repellency was reduced in a pyrethroid-resistant strain that carries two mutations in the sodium channel gene that are responsible for knockdown resistance (kdr) to pyrethroids, indicating a role of activation of sodium channels in bioallethrin repellency. Results from this study show that bioallethrin repellency is likely to be the result of co-activation of Or(s) and sodium channels. These findings not only contribute to our understanding of the modes of action of volatile pyrethroids in spatial repellency, but also provide a framework for developing new repellents based on the dual-target mechanism revealed. © 2021 Society of Chemical Industry.

Olfaction-Related Gene Expression in the Antennae of Female Mosquitoes From Common Aedes aegypti Laboratory Strains.

Adult female mosquitoes rely on olfactory cues like carbon dioxide and other small molecules to find vertebrate hosts to acquire blood. The molecular physiology of the mosquito olfactory system is critical for their host preferences. Many laboratory strains of the yellow fever mosquito Aedes aegypti have been established since the late 19th century. These strains have been used for most molecular studies in this species. Some earlier comparative studies have identified significant physiological differences between different laboratory strains. In this study, we used a Y-tube olfactometer to determine the attraction of females of seven different strains of Ae. aegypti to a human host: UGAL, Rockefeller, Liverpool, Costa Rica, Puerto Rico, and two odorant receptor co-receptor (Orco) mutants Orco2 and Orco16. We performed RNA-seq using antennae of Rockefeller, Liverpool, Costa Rica, and Puerto Rico females. Our results showed that female Aedes aegypti from the Puerto Rico strain had significantly reduced attraction rates toward human hosts compared to all other strains. RNA-seq analyses of the antenna transcriptomes of Rockefeller, Liverpool, Costa Rica, and Puerto Rico strains revealed distinct differences in gene expression between the four strains, but conservation in gene expression patterns of known human-sensing genes. However, we identified several olfaction-related genes that significantly vary between strains, including receptors with significantly different expression in mosquitoes from the Puerto Rico strain and the other strains.

Commonly Used Insect Repellents Hide Human Odors from Anopheles Mosquitoes.

The mode of action for most mosquito repellents is unknown. This is primarily due to the difficulty in monitoring how the mosquito olfactory system responds to repellent odors. Here, we used the Q-system of binary expression to enable activity-dependent Ca2+ imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii. This system allows neuronal responses to common insect repellents to be directly visualized in living mosquitoes from all olfactory organs, including the antenna. The synthetic repellents N,N-diethyl-meta-toluamide (DEET) and IR3535 did not activate Anopheles odorant receptor co-receptor (Orco)-expressing olfactory receptor neurons (ORNs) at any concentration, and picaridin weakly activated ORNs only at high concentrations. In contrast, natural repellents (i.e. lemongrass oil and eugenol) strongly activated small numbers of ORNs in the Anopheles mosquito antennae at low concentrations. We determined that DEET, IR3535, and picaridin decrease the response of Orco-expressing ORNs when these repellents are physically mixed with activating human-derived odorants. We present evidence that synthetic repellents may primarily exert their olfactory mode of action by decreasing the amount of volatile odorants reaching ORNs. These results suggest that synthetic repellents disruptively change the chemical profile of host scent signatures on the skin surface, rendering humans invisible to Anopheles mosquitoes.

Material stiffness variation in mosquito antennae.

The antennae of mosquitoes are model systems for acoustic sensation, in that they obey general principles for sound detection, using both active feedback mechanisms and passive structural adaptations. However, the biomechanical aspect of the antennal structure is much less understood than the mechano-electrical transduction. Using confocal laser scanning microscopy, we measured the fluorescent properties of the antennae of two species of mosquito—Toxorhynchites brevipalpis and Anopheles arabiensis—and, noting that fluorescence is correlated with material stiffness, we found that the structure of the antenna is not a simple beam of homogeneous material, but is in fact a rather more complex structure with spatially distributed discrete changes in material properties. These present as bands or rings of different material in each subunit of the antenna, which repeat along its length. While these structures may simply be required for structural robustness of the antennae, we found that in FEM simulation, these banded structures can strongly affect the resonant frequencies of cantilever-beam systems, and therefore taken together our results suggest that modulating the material properties along the length of the antenna could constitute an additional mechanism for resonant tuning in these species.

Highly-damped nanofiber mesh for ultrasensitive broadband acoustic flow detection

Many small animals have evolved ultrasensitive hearing capability by detecting acoustic flow with mechanosensory hair-like structures. The highly-damped hairy structures such as the fluffy mosquito antennae are driven to move by viscous forces in the fluctuating medium, in contrast to sensing acoustic pressure with tympanal membranes. Inspired by the acoustic flow sensing of animals, the highly-damped 2D nanofiber mesh is presented here for the ultrasensitive, broadband acoustic flow detection. By evaporating 90 nm Au on a freestanding electrospun Poly(methyl methacrylate) (PMMA) nanofiber mesh, we fabricate a thin layer of conductive Au-PMMA nanomesh with dimensions 8 mm × 8 mm, with thickness of approximately 3 μm and average fiber diameter around 430 nm. The nanomesh is shown to move instantaneously with broadband fluctuating airflow near maximum physical efficiency from 100 Hz to 10 000 Hz, i.e. , surpassing the frequency response and efficiency of previously-reported microscale hairy structures. By transducing the motion of the nanomesh with electromagnetic induction, we demonstrate fluctuating airflow detection with almost full fidelity in the measured frequency range. Sensing flow with highly-damped nanomaterials will benefit miniaturized flow measurement and sound detection, and provides an important application for the diverse flourishing nanomaterials, such as 1D nanofibers, 2D nanomeshes, and 3D nanolattices.

Reverse chemical ecology approach for the identification of an oviposition attractant for Culex quinquefasciatus

Pheromones and other semiochemicals play a crucial role in today's integrated pest and vector management strategies. These semiochemicals are typically discovered by bioassay-guided approaches. Here, we applied a reverse chemical ecology approach; that is, we used olfactory proteins to lead us to putative semiochemicals. Specifically, we used 7 of the top 10 odorant receptors (ORs) most expressed in the antennae of the southern house mosquito, Culex quinquefasciatus, and which are yet to be deorphanized. We expressed these receptors in the Xenopus oocyte recording system and challenged them with a panel of 230 odorants, including physiologically and behaviorally active compounds. Six of the ORs were silent either because they are not functional or a key odorant was missing. CquiOR36, which showed the highest transcript levels of all OR genes in female antennae, was also silent to all odorants in the tested panel, but yielded robust responses when it was accidentally challenged with an old sample of nonanal in ethanol. After confirming that fresh samples were inactive and through a careful investigation of all possible "contaminants" in the old nonanal samples, we identified the active ligand as acetaldehyde. That acetaldehyde is activating CquiOR36 was further confirmed by electroantennogram recordings from antennae of fruit flies engineered to carry CquiOR36. Antennae of female mosquitoes also responded to acetaldehyde. Cage oviposition and dual-choice assays demonstrated that acetaldehyde is an oviposition attractant in a wide range of concentrations and thus of potential practical applications.

In Silico Site-Directed Mutagenesis of the Anopheles gambiae Odorant Binding Protein 20

Abstract The Anopheles gambiae is a highly anthropophilic mosquito which is the leading vector for malaria. This disease has affected more than 500 million people worldwide. The Anopheles gambiae targets its hosts through the odors of the human skin and sweat where odorant molecules radiate. These odors elicit specific responses from the insect through the odorant – binding proteins (OBP). Recently, a specific type of OBP has been characterized which is known as the Anopheles gambiae odorant – binding protein 20 (AgamOBP20). This OBP is highly expressed in the female mosquito antennae during the peak of its host – seeking behavior and thus may play a role in olfactory perception. The binding site of the AgamOBP20 is composed primarily of hydrophobic residues wherein the importance of each residue is herein analysed to further understand the properties of AgamOBP20. This was carried out through computer – aided site – directed mutagenesis coupled with homology modelling and docking simulations wherein each residue in the binding site was changed to alanine and serine. Probable key amino acid residues were identified as LEU106, LEU107, and MET53 which are hypothesized to play a significant role in the protein – ligand interaction. These residues had the greatest impact in the binding free energy when mutated with alanine and serine. The presented results suggest that steric hindrance and hydrophobic interaction are crucial factors to consider on the manner in which the ligand binds with AgamOBP20. The molecular features and parameters obtained may be utilized for the development of new pesticides and repellents that are able to block the function of AgamOBP20 and may result to the disarray of the host – seeking behavior of the Anopheles gambiae.

Directional hearing: from biophysical binaural cues to directional hearing outdoors.

When insects communicate by sound, or use acoustic cues to escape predators or detect prey or hosts they have to localize the sound in most cases, to perform adaptive behavioral responses. In the case of particle velocity receivers such as the antennae of mosquitoes, directionality is no problem because such receivers are inherently directional. Insects equipped with bilateral pairs of tympanate ears could principally make use of binaural cues for sound localization, like all other animals with two ears. However, their small size is a major problem to create sufficiently large binaural cues, with respect to both interaural time differences (ITDs, because interaural distances are so small), but also with respect to interaural intensity differences (IIDs), since the ratio of body size to the wavelength of sound is rather unfavorable for diffractive effects. In my review, I will only shortly cover these biophysical aspects of directional hearing. Instead, I will focus on aspects of directional hearing which received relatively little attention previously, the evolution of a pressure difference receiver, 3D-hearing, directional hearing outdoors, and directional hearing for auditory scene analysis.