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.
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.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Display omitted
•Odor responses of An. coluzzii ORNs revealed by a genetically encoded calcium sensor•Natural repellents activate olfactory receptor neurons•Synthetic repellents likely do not activate Anopheles olfactory receptor neurons•Synthetic repellents reduce the amount of odorants reaching the antenna
The olfactory mode of action for mosquito repellents is unclear. Afify et al. genetically engineered Anopheles mosquitoes to reveal odor-induced activities in olfactory neurons. Natural repellents activated olfactory neurons, and synthetic repellents did not. Synthetic repellents instead likely reduce attraction to humans by decreasing the amount of odorants reaching the mosquito.
olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in
, 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:
for ORs, and
,
, and
for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in
(
). 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,
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.
,
, and
expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing
mutant antennal and palpal neurons identify changes in olfactory responses. We also find co-expression of
and
in
and
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.
Catnip (Nepeta cataria) is a common garden herb well known for its euphoric and hallucinogenic effects on domestic cats,1–3 for its medicinal properties,4,5 as well as for its powerful repellent ...action on insects.6,7 Catnip extracts have been proposed as a natural alternative to synthetic insect repellents, such as N,N-diethyl-3-methylbenzamide (DEET),8,9 but how catnip triggers aversion in insects is not known. Here, we show that, both in Drosophila melanogaster flies and Aedes aegypti mosquitoes, the major mediator of catnip repellency is the widely conserved chemical irritant receptor TRPA1. In vitro, both catnip extract and its active ingredient nepetalactone can directly activate fly and mosquito TRPA1. In vivo, D. melanogaster and Ae. aegypti TRPA1 mutants are no longer repelled by catnip and nepetalactone. Interestingly, our data show that some, but not all, fly and mosquito TRPA1 variants are catnip targets. Moreover, unlike the broad TRPA1 agonist allyl isothiocyanate (AITC) (an active ingredient of tear gas and wasabi), catnip does not activate human TRPA1. Our results support the use of catnip and nepetalactone as insect-selective irritants and suggest that, despite TRPA1’s broad conservation, insect TRPA1 can be targeted for the development of safe repellents.
Display omitted
•Catnip is a broad-acting insect repellent•The repellency is mediated via iridoids such as nepetalactone•Catnip and nepetalactone activate select insect TRPA1 isoforms•TRPA1-deficient mosquitoes show no aversion to catnip
Catnip has been used for millennia as an insect repellent. Melo et al. find that catnip and its major iridoid component nepetalactone activate insect isoforms of the irritant receptor TRPA1. Mosquitoes lacking TRPA1 are no longer repelled by catnip. Catnip does not activate human TRPA1, and this supports its use as a safe natural mosquito repellent.
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.
Display omitted
•Ectopic OR expression disrupts mosquito olfactory neuron activity•Expression of an OR in mosquitoes reduces endogenous OR expression•Onset of mosquito OR expression extends into adult stages•Mosquitoes host seek despite the lack of OR neuron function
Maguire et al. discover that the ectopic expression of an olfactory receptor can downregulate the transcription of endogenous odorant receptors in mosquito olfactory neurons. The onset of mosquito odorant-receptor expression by an olfactory neuron continues into adult stages, and is particularly sensitive to exogenous olfactory reception expression.
Methyl N,N-dimethyl anthranilate (MDA), ethyl anthranilate (EA) and butyl anthranilate (BA) were previously shown to repel Aedes aegypti mosquitoes from landing on human skin. However, the effect of ...these compounds on the orientation of flying mosquitoes in a choice situation and their effect on mosquito oviposition are not yet known. Here, we used a modified Y-tube olfactometer to test the effect of these compounds on the orientation of Aedes aegypti flying towards skin odor (human fingers), and we tested their effect on Aedes aegypti oviposition choice in a cage assay. In both behavioral situations we compared the effect to the well-documented repellent N,N-diethyl-meta-toluamide (DEET). MDA, EA, and DEET inhibited Aedes aegypti from flying towards skin odor while BA had no such effect. Conversely, MDA had no effect on oviposition while EA, BA, and DEET deterred oviposition, with the strongest effect observed for BA. Thus, we confirm that EA and DEET are generally repellent, while MDA is repellent only in a host-seeking context, and BA is deterrent only in an oviposition context. These compounds appear of potential use in mosquito control programs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Fruit flies recognize hundreds of ecologically relevant odors and respond appropriately to them. The complexity, redundancy and interconnectedness of the olfactory machinery complicate efforts to ...pinpoint the functional contributions of any component neuron or receptor to behavior. Some contributions can only be elucidated in flies that carry multiple mutations and transgenes, but the production of such flies is currently labor-intensive and time-consuming. Here, we describe a set of transgenic flies that express the
GAL80 in specific olfactory sensory neurons (
). The GAL80s effectively and specifically subtract the activities of GAL4-driven transgenes that impart anatomical and physiological phenotypes.
can allow researchers to efficiently activate only one or a few types of functional neurons in an otherwise nonfunctional olfactory background. Such experiments will improve our understanding of the mechanistic connections between odorant inputs and behavioral outputs at the resolution of only a few functional neurons.
BACKGROUND: p-cresol (4-methylphenol) and its isomer m-cresol (3-methylphenol) have been shown to activate the same sensilla in Aedes aegypti (Linnaeus) mosquitoes. Whereas p-cresol has been ...suggested to play a role in oviposition site choice, the behavioral significance of m-cresol is unknown. METHODS: Here, we assayed the oviposition behavior of Aedes aegypti towards p-cresol and m-cresol using cage assay. Specifically we tested different concentrations of p-cresol (10⁻¹²-10³ ppm) and m-cresol (10⁻¹-10³ ppm), the 1:1 mixture of the two compounds at 10² ppm, and the two individual compounds at 10² ppm together in the same cage. RESULTS: We show that (1) p-cresol is a stimulant at a low concentration and deterrent over a broad range of higher concentrations (10⁻⁸-10³ ppm), while m-cresol was behaviorally ineffective, except for a deterrent effect at the highest concentration (10³ ppm) (2) in concentration choice tests (different concentrations tested against each other), both compounds were deterrent only at the highest concentration (3) a 1:1 mixture of both compounds exhibited a deterrent effect on oviposition (4) when presented in separate cups but together in the same cage, p-cresol and m-cresol (10² ppm) both received significantly less eggs than water alone. CONCLUSIONS: Our results suggest that p-cresol is a strong oviposition deterrent with a stimulant effect at only a very low concentration, while m-cresol is not a deterrent per se. However, in the presence of p-cresol in the vicinity, m-cresol acts as a deterrent. This finding adds a new twist to the possible interactions of different odors in oviposition site choice: not only the source itself, but nearby odors also influence a mosquito’s choice.
Gravid mosquitoes use chemosensory (olfactory, gustatory, or both) cues to select oviposition sites suitable for their offspring. In nature, these cues originate from plant infusions, microbes, ...mosquito immature stages, and predators. While attractants and stimulants are cues that could show the availability of food (plant infusions and microbes) and suitable conditions (the presence of conspecifics), repellents and deterrents show the risk of predation, infection with pathogens, or strong competition. Many studies have addressed the question of which substances can act as positive or negative cues in different mosquito species, with sometimes apparently contradicting results. These studies often differ in species, substance concentration, and other experimental details, making it difficult to compare the results. In this review, we compiled the available information for a wide range of species and substances, with particular attention to cues originating from larval food, immature stages, predators, and to synthetic compounds. We note that the effect of many substances differs between species, and that many substances have been tested in few species only, revealing that the information is scattered across species, substances, and experimental conditions.
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,
, 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
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).
PDF
