Repellents Inhibit P450 Enzymes in Stegomyia (Aedes) aegypti

The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s) underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES) arm-in cage assay with Stegomyia (Aedes) aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils.     

Rhodnius prolixus smells repellents: Behavioural evidence and test of present and potential compounds inducing repellency in Chagas disease vectors

Insect repellents are known since many decades ago and constitute a major tool for personal protection against the biting of mosquitoes. Despite their wide use, the understanding of why and how repellents repel is relatively recent. In particular, the question about to what extent insects other than mosquitoes are repulsed by repellents remains open. We developed a series of bioassays aimed to test the performance of well established as well as potential repellent molecules on the Chagas disease vector Rhodnius prolixus. Besides testing their ability to prevent biting, we tested the way in which they act, i.e., by obstructing the detection of attractive odours or by themselves. By using three different experimental protocols (host-biting, open-loop orientation to odours and heat-triggered proboscis extension response) we show that DEET repels bugs both in the presence and in the absence of host-associated odours but only at the highest quantities tested. Piperidine was effective with or without a host and icaridine only repelled in the absence of a living host. Three other molecules recently proposed as potential repellents due to their affinity to the Ir40a⁺ receptor (which is also activated by DEET) did not evoke significant repellency. Our work provides novel experimental tools and sheds light on the mechanism behind repellency in haematophagous bugs.     

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.     

Efficacy of the new repellent BioUD<Superscript>®</Superscript> against three species of ixodid ticks

BioUD ^® with the active ingredient 2-undecanone originally derived from wild tomato plants is a new repellent recently registered by the US EPA. Repellent efficacy of BioUD ^® (7.75% 2-undecanone) and DEET (98.11%) was examined in the laboratory using a choice test between repellent-treated and control filter paper surfaces for Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. BioUD ^® provided greater repellency against A. americanum and I. scapularis than DEET. No difference was found between BioUD ^® and DEET against D. variabilis. In head-to-head assays between BioUD ^® and DEET, undiluted and 50% dilutions of BioUD^® were more repellent than undiluted DEET against all three species tested. Similarly, a 25% dilution of BioUD^® was more repellent than DEET against A. americanum while no difference in mean percentage repellency was found between a 25% dilution of BioUD^® and DEET against I. scapularis. Based on regression analysis, the concentration of BioUD^® required for equivalent repellency to 98.11% DEET was 39.5% for D. variabilis and 29.7% for I. scapularis. A log-probit model could not be constructed for A. americanum from the dosages tested. Based on filter paper head-to-head assays, BioUD^® is at least 2–4 times more active as a repellent than DEET against three species of ixodid ticks under the conditions of our laboratory bioassays.     

The mysterious multi-modal repellency of DEET

DEET is the most effective insect repellent available and has been widely used for more than half a century. Here, I review what is known about the olfactory and contact mechanisms of DEET repellency. For mosquitoes, DEET has at least two molecular targets: Odorant Receptors (ORs) mediate the effect of DEET at a distance, while unknown chemoreceptors mediate repellency upon contact. Additionally, the ionotropic receptor Ir40a has recently been identified as a putative DEET chemosensor in Drosophila. The mechanism of how DEET manipulates these molecular targets to induce insect avoidance in the vapor phase is also contested. Two hypotheses are the most likely: DEET activates an innate olfactory neural circuit leading to avoidance of hosts (smell and avoid hypothesis) or DEET has no behavioral effect on its own, but instead acts cooperatively with host odors to drive repellency (confusant hypothesis). Resolving this mystery will inform the search for a new generation of insect repellents.     

Different Repellents for Aedes aegypti against Blood-Feeding and Oviposition

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.     

Optimization of pyrethroid and repellent on fabrics against Stegomyia albopicta (=Aedes albopictus) using a microencapsulation technique

A new approach employing a combination of pyrethroid and repellent is proposed to improve the protective efficacy of conventional pyrethroid‐treated fabrics against mosquito vectors. In this context, the insecticidal and repellent efficacies of commonly used pyrethroids and repellents were evaluated by cone tests and arm‐in‐cage tests against Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae). At concentrations of LD₅₀ (estimated for pyrethroid) or ED₅₀ (estimated for repellent), respectively, the knock‐down effects of the pyrethroids or repellents were further compared. The results obtained indicated that deltamethrin and DEET were relatively more effective and thus these were selected for further study. Synergistic interaction was observed between deltamethrin and DEET at the ratios of 5 : 1, 2 : 1, 1 : 1 and 1 : 2 (but not 1 : 5). An optimal mixing ratio of 7 : 5 was then microencapsulated and adhered to fabrics using a fixing agent. Fabrics impregnated by microencapsulated mixtures gained extended washing durability compared with those treated with a conventional dipping method. Results indicated that this approach represents a promising method for the future impregnation of bednet, curtain and combat uniform materials.     

An important role of a pyrethroid-sensing residue F1519 in the action of the N-alkylamide insecticide BTG 502 on the cockroach sodium channel

Deltamethrin, a pyrethroid insecticide, and BTG 502, an alkylamide insecticide, target voltage-gated sodium channels. Deltamethrin binds to a unique receptor site and causes prolonged opening of sodium channels by inhibiting deactivation and inactivation. Previous ²²Na⁺ influx and receptor binding assays using mouse brain synaptoneurosomes showed that BTG 502 antagonized the binding and action of batrachotoxin (BTX), a site 2 sodium channel neurotoxin. However, the effect of BTG 502 has not been examined directly on sodium channels expressed in Xenopus oocytes. In this study, we examined the effect of BTG 502 on wild-type and mutant cockroach sodium channels expressed in Xenopus oocytes. Toxin competition experiments confirmed that BTG 502 antagonizes the action of BTX and possibly shares a common receptor site with BTX. However, unlike BTX which causes persistent activation of sodium channels, BTG 502 reduces the amplitude of peak sodium current. A previous study showed that BTG 502 was more toxic to pyrethroid-resistant house flies possessing a super-kdr (knockdown resistance) mechanism than to pyrethroid-susceptible house flies. However, we found that the cockroach sodium channels carrying the equivalent super-kdr mutations (M918T and L1014F) were not more sensitive to BTG 502 than the wild-type channel. Instead, a kdr mutation, F1519I, which reduces pyrethroid binding, abolished the action of BTG 502. These results provide evidence the actions of alkylamide and pyrethroid insecticides require a common sodium channel residue.     

Laboratory Evaluation of Controlled-release Repellent Treated Pulp Fabric on Human Volunteers Against Mosquito Vectors

Three controlled‐release personal‐use pulp fabric impregnated insect repellent formulations of 5% N, N‐diethyl‐3‐methylbenzamide (DEET), and 10% and 15% neem oils were evaluated in an environmental chamber on volunteers for their repellent efficacy against three mosquito species, Culex pipiens pallens, Aedes aegypti and Ochlerotatus togoi. The 5% DEET formulation showed significant repellency in pulp fabric (5 mm in width) against Culex pipiens pallens and Aedes aegypti (P < 0.05), providing an average repellency of 88.0%, 66.3%, and 46.8% of Cx. pipiens pallens, Ae. Aegypti and O. togoi bites, respectively, during the 6 hours of exposure period. Against night‐biting mosquitoes Cx. pipiens pallens, the DEET formulation provided mostly complete protection for at least 4 hours after the application. In pulp fabric of 10 mm in width, the 5% deet formulation showed significantly the highest repellency among the repellents against O. togoi (P<0.05), providing an average repellency of 52.3% during the 6 hours of exposure period. However, the pulp fabrics treated with 10% and 15% neem oil were less effective than 5% DEET against three mosquito species. This study demonstrated the potential of 5% DEET as pulp fabric repellent against both day‐ and night‐biting mosquitoes.     

Toxicity of pyrethroids and repellency of diethyltoluamide in two deltamethrin-resistant colonies of Triatoma infestans Klug, 1834 (Hemiptera: Reduviidae)

The aim of the currrent investigation was to evaluate (a) the toxicity of three pyrethroids (deltamethrin, lambda-cyhalothrin, and tetramethrin); (b) the effect of these insecticides on the locomotor activity; and (c) the repellent effect of N,N-diethyl-m-toluamide (DEET) on two deltamethrin-resistant strains of Triatoma infestans from Argentina (El Chorro and La Toma), and one susceptible strain. The resistance ratios (RRs) obtained for the La Toma strain were: > 10,769, 50.7, and > 5.2 for deltamethrin, lambda-cyhalothrin, and tetramethrin respectively. The RRs for the El Chorro strain were: > 10,769, 85.8, and > 5.2 for deltamethrin, lambda-cyhalothrin, and tetramethrin respectively. The hyperactivity usually caused by the three pyrethroids was in both the deltamethrin-resistant strains compared to the susceptible reference strain. No differences were observed in the repellent effect of DEET between the three groups. These results indicate that the deltamethrin-resistant insects have a cross resistance to lambda-cyhalothrin and tetramethrin, and are also resistant to the first symptom of pyrethroid poisoning (hyperactivity). However, the sensorial process related to DEET repellency does not appear to be altered.     

Sustained efficacy of the novel topical repellent TT‐4302 against mosquitoes and ticks

Mosquitoes and ticks are blood‐feeding pests of humans and animals that can vector many important aetiological agents of disease. Previous research demonstrated that TT‐4302 (Guardian^® Wilderness) containing 5% geraniol applied to human subjects gave 5–6 h of repellency against mosquitoes (depending on species) and was repellent to ticks in vitro. This study was conducted to obtain an independent third‐party evaluation of TT‐4302 against Stegomyia aegypti (= Aedes aegypti) (Diptera: Culicidae) mosquitoes and to test the efficacy of the product in the field against ticks. TT‐4302 provided an average of 6.5 h of repellency of ≥ 95% [Weibull mean protection time: 7.4 h, 95% confidence interval (CI) 5.8–11.3 h] for St. aegypti, whereas a 15% DEET formulation provided 4.7 h of repellency (Weibull mean protection time: 5.2 h, 95% CI 3.7–6.9 h). In tick field trials, the efficacy of TT‐4302 did not differ significantly from that of a 25% DEET formulation against Amblyomma americanum (Ixodida: Ixodidae). TT‐4302 was 81.3% repellent at 2.5 h after application, whereas DEET was 77.2% repellent at the same time‐point. Results at 3.5 h after application were 71.4% for TT‐4302 and 72.9% for DEET.     

Repellency of essential oils and their components to the human body louse, Pediculus humanus humanus

Five essential oils and nine of their components were compared to diethyl toluamide (DEET) for their repellent activity against the human body louse, Pediculus humanus humanus. The absolute or intrinsic repellency of the compounds was tested by applying the repellent to corduroy patches and comparing them with untreated patches. It was found that the most effective repellents were DEET and citronella, whose activity lasted at least 29 days. The activity of rosemary lasted at least 18 days and that of eucalyptus more than 8 days. The repellent activity of the oil components such as citronellal and geraniol lasted more than 15 and 8 days, respectively. DEET remained effective at a dilution of 1:32, geraniol at 1:8, citronella at 1:4 and rosemary and citronellal at 1:1. The comparative or standard repellency of the candidate repellents was examined with the aid of a new screening technique using hairs treated with ammonium bicarbonate which is attractive to lice. Using this technique it could be shown that the repellent activity of citronella and geraniol lasted 2 days and that of rosemary and citronellal for only one day. DEET was active for less than one day. Serial dilutions of these substances also revealed that citronella was the most potent repellent for lice, followed by citronellal, rosemary, geraniol and DEET. The differences however, were not significant.     

Locomotor Behaviour of Blattella germanica Modified by DEET

N,N-diethyl-3-methylbenzamide (DEET) is the active principle of most insect repellents used worldwide. However, its toxicity on insects has not been widely studied. The aim of this work is to study the effects of DEET on the locomotor activity of Blattella germanica. DEET has a dose-dependent repellent activity on B. germanica. Locomotor activity was significantly lower when insects were pre-exposed to 700 µg/cm² of DEET for 20 or 30 minutes, but it did not change when pre-exposure was shorter. Locomotor activity of insects that were pre-exposed to 2.000 µg/cm² of DEET for 10 minutes was significantly lower than the movement registered in controls. No differences were observed when insects were pre-exposed to lower concentrations of DEET. A 30-minute pre-exposure to 700 µg/cm² of DEET caused a significant decrease in locomotor activity. Movement was totally recovered 24 h later. The locomotor activity measured during the exposure to different concentrations of DEET remained unchanged. Insects with decreased locomotor activity were repelled to the same extent than control insects by the same concentration of DEET. We demonstrated that the repellency and modification of locomotor activity elicited by DEET are non-associated phenomena. We also suggested that the reduction in locomotor activity indicates toxicity of DEET, probably to insect nervous system.     

Repellency of synthetic and plant-derived preparations for Culicoides imicola

Abstract The study was conducted to find a safer and longer lasting repellent for C. imicola than di-ethyl toluamide (DEET), to validate whether the current recommendations in Israel for application of repellents during an outbreak of disease caused by pathogens borne by Culicoides imicola are justified, and to test plant-derived preparations as potential replacements for the synthetic repellents. Seven repellents were compared by a method using treated netting across the entrance of a suction light trap. Those inferior to DEET were: oregano and Herbipet ® which showed a slight non-significant repellency for 2 h, Tri-Tec14 ™ which showed significant (P < 0.05) repellency with respect to controls for 2h and Stomoxin ® which showed significant (P < 0.05) repellency for lh. As the active ingredient of Stomoxin is permethrin, this indicates that recommendations to spray animals with this insecticide to prevent the spread of C.imico/a-borne pathogens will not be useful. The repellents superior to DEET were: the plant-derived material AglOOO that repelled significantly (P < 0.05) with respect to controls for up to 4h following a similar pattern to but somewhat more strongly than DEET, and pyrethroid-T which exerted significant (P < 0.05) repellency for 9h. Pyrethroid-T proved to be the best repellent tested, and if sprayed nightly it might provide protection from C. imicola-bome pathogens.     

The mysterious multi-modal repellency of DEET

DEET is the most effective insect repellent available and has been widely used for more than half a century. Here, I review what is known about the olfactory and contact mechanisms of DEET repellency. For mosquitoes, DEET has at least two molecular targets: Odorant Receptors (ORs) mediate the effect of DEET at a distance, while unknown chemoreceptors mediate repellency upon contact. Additionally, the ionotropic receptor Ir40a has recently been identified as a putative DEET chemosensor in Drosophila. The mechanism of how DEET manipulates these molecular targets to induce insect avoidance in the vapor phase is also contested. Two hypotheses are the most likely: DEET activates an innate olfactory neural circuit leading to avoidance of hosts (smell and avoid hypothesis) or DEET has no behavioral effect on its own, but instead acts cooperatively with host odors to drive repellency (confusant hypothesis). Resolving this mystery will inform the search for a new generation of insect repellents.     

Diurnal test periods influence behavioral responses of Aedes aegypti (Diptera: Culicidae) to repellents

There is a broad understanding of the influence of environmental factors on various aspects of normal mosquito behavior. How these external factors influence responses to repellent compounds is far less clear. The objective of this study was to investigate the effect of different daytime periods combining the normal circadian activity of a laboratory colony of Aedes aegypti (L.) with behavioral responses of mosquitoes exposed to three different compounds possessing repellent properties. Using an excito-repellency test chamber with different test designs (contact irritancy + repellency and noncontact repellency), female mosquitoes were exposed to each chemical or matching blank control during four different 3-h time intervals beginning 0600 to 1800?h. Mosquitoes showed more significant avoidance responses (escape movement away from the chemical) when exposed to either DEET or hairy basil during the afternoon periods. With deltamethrin, there was no significant difference in repellent escape movement during any period of testing. Escape activity with deltamethrin was significantly greater during all diurnal periods in contact tests compared to DEET and hairy basil. From this study, it was shown that time of diurnal testing can significantly influence behavioral responses of Ae. aegypti exposed to chemical-based repellents. Therefore, the assessment of chemicals (toxins, repellents, attractants) and must carefully consider time-of-test as a potential confounding factor during evaluation and comparisons.     

Functional Expression of Drosophila para Sodium Channels : Modulation by the Membrane Protein TipE and Toxin Pharmacology

The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (K _d ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.     

Novel field assays and the comparative repellency of BioUD(®) , DEET and permethrin against Amblyomma americanum

Two new field bioassay methods were developed to compare the repellent activity of BioUD^® (containing 7.75% 2‐undecanone), 98.1% DEET and 0.5% permethrin against natural populations of nymphal Amblyomma americanum (L.) (Acari: Ixodidae). In a cloth sheet assay, pieces of material measuring 41 × 58 cm, separately treated with one of the test materials or the appropriate solvent carrier, were placed at random on the ground and baited with dry ice for 1 h. Mean numbers of ticks on repellent‐treated sheets were significantly lower than on control sheets. There was no significant difference in the number of ticks collected between sheets treated with BioUD^® and those treated with DEET. However, significantly fewer ticks were found on sheets treated with BioUD^® or DEET than on permethrin‐treated sheets. In a sock test, over‐the‐calf tube socks were treated with one of the test materials or the appropriate solvent carrier. Human volunteers wore a repellent‐treated and a corresponding carrier‐treated sock on either leg and walked randomly over an area of approximately 4000 m² for 15 min. Significantly fewer ticks were collected from socks treated with BioUD^® or DEET than from socks treated with the carrier and there was no significant difference in repellency between these two agents. No difference in the mean number of ticks collected was found between permethrin‐treated and corresponding carrier‐treated socks. To examine the mechanism of repellency of BioUD^®, a four‐choice olfactometer was used to assess spatial repellency against adult A. americanum. As expected in the absence of a repellent, when all choices were represented by water‐treated filter paper, ticks were equally distributed among the choices. When one choice consisted of BioUD^®‐treated filter paper and the remaining choices of water‐treated paper, the distribution of ticks on the repellent‐treated paper was significantly lower than might be expected to occur by chance, suggesting that repellency is at least partly achieved by an olfactory mechanism.     

Synergy between Repellents and Organophosphates on Bed Nets: Efficacy and Behavioural Response of Natural Free-Flying An. gambiae Mosquitoes

Background

Chemicals are used on bed nets in order to prevent infected bites and to kill aggressive malaria vectors. Because pyrethroid resistance has become widespread in the main malaria vectors, research for alternative active ingredients becomes urgent. Mixing a repellent and a non-pyrethroid insecticide seemed to be a promising tool as mixtures in the laboratory showed the same features as pyrethroids.

Methodology/Principal Findings

We present here the results of two trials run against free-flying Anopheles gambiae populations comparing the effects of two insect repellents (either DEET or KBR 3023, also known as icaridin) and an organophosphate insecticide at low-doses (pirimiphos-methyl, PM) used alone and in combination on bed nets. We showed that mixtures of PM and the repellents induced higher exophily, blood feeding inhibition and mortality among wild susceptible and resistant malaria vectors than compounds used alone. Nevertheless the synergistic interactions are only involved in the high mortality induced by the two mixtures.

Conclusion

These field trials argue in favour of the strategy of mixing repellent and organophosphate on bed nets to better control resistant malaria vectors.