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Background: 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., Results: 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., Conclusions: 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., (© 2024 Society of Chemical Industry.)

Volatile pyrethroids are effective in reducing mosquito populations and repelling vectors away from hosts. However, many gaps in knowledge exist for the sublethal impacts of volatile pyrethroids on mosquitoes. To that end, transfluthrin exposures were conducted on a field strain of Aedes albopictus (Skuse) held as a laboratory colony. Dose-response analysis was conducted on both sexes at either 1-4 days old or 5-10 days old. Resultant concentration data were used to evaluate the LC20 and LC50 values in various mate pairings of treatments and controls in which either the male or female was from a selectively treated group and mated with a counterpart that was treated independently. Blood feeding proportion, delayed mortality after a 24-h recovery period, egg collection totals, and F1 larval survival were determined following transfluthrin treatment in the F0, but outcomes were not significant. In contrast, sterility was predicated on male treatment, with treated females resulting in higher overall egg viability. Treated males in the mating pair resulted in significantly lower egg viability and accelerated larval hatch in the F1. Additionally, the presence of sperm in female spermathecae was significantly diminished in test groups containing treated male mosquitoes. Male sublethal effects may be a critical determinant of a mixed population's reproductive success., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The dinoflagellate Karenia brevis is a causative agent of red tides in the Gulf of Mexico and generates a potent family of structurally related brevetoxins that act via the voltage-sensitive Na + channel. This project was undertaken to better understand the neurotoxicology and kdr cross-resistance to brevetoxins in house flies by comparing the susceptible aabys strain to ALkdr (kdr) and JPskdr (super-kdr). When injected directly into the hemocoel, larvae exhibited rigid, non-convulsive paralysis consistent with prolongation of sodium channel currents, the known mechanism of action of brevetoxins. In neurophysiological studies, the firing frequency of susceptible larval house fly central nervous system preparations showed a > 200% increase 10 min after treatment with 1 nM brevetoxin-3. This neuroexcitation is consistent with the spastic paralytic response seen after hemocoel injections. Target site mutations in the voltage-sensitive sodium channel of house flies, known to confer knockdown resistance (kdr and super-kdr) against pyrethroids, attenuated the effect of brevetoxin-3 in baseline firing frequency and toxicity assays. The rank order of sensitivity to brevetoxin-3 in both assays was aabys > ALkdr > JPskdr. At the LD 50 level, resistance ratios for the knockdown resistance strains were 6.9 for the double mutant (super-kdr) and 2.3 for the single mutant (kdr). The data suggest that knockdown resistance mutations may be one mechanism by which flies survive brevetoxin-3 exposure during red tide events., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: An important component of the biological activity of pyrethroids, when used in disease vector control, is excito-repellency. In this study, behavioral differences between insecticide susceptible (Orlando) and pyrethroid resistant (Puerto Rican) strains of Aedes aegypti were explored in a round glass arena using fabrics treated with permethrin, etofenprox, deltamethrin, or DDT. Repellency was evaluated across several variables, including the time to first flight (TFF), number of landings (NOL), total flight time (TFT), and maximum surface contact (MSC), all by video analysis., Results: Results from the Orlando strain indicated they were less likely than the Puerto Rican strain to tolerate tarsal contact with the treated fabrics. All four response variables indicated that the mosquito flight and landing behavior was most affected by pyrethroid resistance [knockdown resistance (kdr)] status. In other experiments, mosquitoes were surgically altered, with antennae ablated bilaterally, and these mosquitoes were more likely to stay on the treated surfaces for longer periods of time, irrespective of any chemical exposure. There were also differences in the responses to antennal ablation between the two strains of mosquitoes, indicating that resistance factors, probably kdr, influence the reactivity of mosquitoes to pyrethroid and DDT treatments, and that it was not completely negated by antennal ablation., Conclusions: These findings confirm the role of antennal olfactory components in the expression of excito-repellent behaviors, and also support the hypothesis that excito-repellency from pyrethroid/DDT exposure is probably due to a combination of sublethal neurotoxic excitation and interactions with the olfactory system. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Chemical screening efforts recently found that 3-phenoxybenzaldehyde, a breakdown product of alpha-cyano pyrethroids, was a potent spatial repellent against Aedes aegypti mosquitoes in a glass tube repellency assay. In order to characterize this molecule further and identify structure-activity relationships, a set of 12 benzaldehyde analogues were screened for their repellency and toxicity in vapor phase exposures at 100 μg/cm 2 . Dose-response analyses were performed for the most active compounds in order to better characterize their repellent potency and toxicity compared to those of other commercially available toxicants. The three most toxic compounds (LC 50 values) were 3-chlorobenzaldehyde (CBA) (37 μg/cm 2 ), biphenyl-3-carboxaldehyde (BCA) (48 μg/cm 2 ), and 3-vinylbenzaldehyde (66 μg/cm 2 ), which makes them less toxic than bioallethrin (6.1 μg/cm 2 ) but more toxic than sandalwood oil (77 μg/cm 2 ), a repellent/toxic plant essential oil. The most repellent analogues with EC 50 values below 30 μg/cm 2 were 3-phenoxybenzaldehyde (6.3 μg/cm 2 ), isophthalaldehyde (23 μg/cm 2 ), BCA (17 μg/cm 2 ), and CBA (22 μg/cm 2 ), which makes them about as active as N,N -diethyl-3-methylbenzamide (25.4 μg/cm 2 ). We further investigated the activity of a select group of these benzaldehydes to block the firing of the central nervous system of A. aegypti larvae. Compounds most capable of repelling and killing mosquitoes in the vapor phase were also those most capable of blocking nerve firing in the larval mosquito nervous system. The results demonstrate that benzaldehyde analogues are viable candidate repellent and insecticidal molecules and may lead to the development of future repellent and vapor toxic vector control tools.

The emergence of pyrethroid-resistant mosquitoes is a worldwide problem that necessitates further research into the development of new repellents and insecticides. This study explored the modification of existing pyrethroid acids to identify structural motifs that might not be affected by kdr active site mutations that elicit pyrethroid resistance. Because synthetic pyrethroids almost always contain activity-dependent chiral centers, we chose to focus our efforts on exploring alkoxy moieties of esters obtained with 1 R - trans -permethrinic and related acids, which we showed in previous studies to have repellent and/or repellent synergistic properties. To this end, compounds were synthesized and screened for spatially acting repellency and insecticidal activity against the susceptible, Orlando, and pyrethroid-resistant, Puerto Rico, strains of Aedes aegypti mosquito. Screening utilized a high-throughput benchtop glass tube assay, and the compounds screened included a mixture of branched, unbranched, aliphatic, halogenated, cyclic, non-cyclic, and heteroatom-containing esters. Structure-activity relationships indicate that n -propyl, n -butyl, n -pentyl, cyclobutyl, and cyclopentyl substituents exhibited the most promising repellent activity with minimal kdr cross resistance. Preliminary testing showed that these small alcohol esters can be synergistic with phenyl amides and pyrethroid acids. Further derivatization of pyrethroid acids offer an interesting route to future active compounds, and while mosquitoes were the focus of this work, pyrethroid acids and esters have potential for use in reducing pest populations and damage in cropping systems as well.

Because of the increased interest in plant essential oils (PEO) for both home pest control and personal bite protection, the ability of fir needle (Abies balsamea) oil to synergize the 1-h knockdown and 24-h toxicity of 9 different synthetic insecticides was evaluated. Fir needle oil strongly synergized knockdown of the neonicotinoids, clothianidin, and thiamethoxam (between 16- and 24-fold), as well as natural pyrethrins (12-fold), but had less effect with organophosphates and fipronil. For 24-h mortality, only pirimiphos-methyl was strongly synergized by fir needle oil pretreatment (18-fold). Chemical analysis and testing identified delta-3-carene is the most bioactive constituent, producing synergism similar to that of the whole oil. In fact, this constituent synergized the 24-h mortality of clothianidin to a higher degree than fir needle oil itself (4.9-fold vs. 2.4-fold). Synergism is unlikely to be mediated by effects on the nervous system, as fir needle oil caused no change in mosquito central nervous system firing at 100 ppm and did not synergize an inactive concentration of natural pyrethrins (10 nM). To better understand fir needle oil effects, we evaluated the ability of pretreatment with this oil to impact Aedes aegypti monooxygenase degradation of a model substrate, 7-ethoxycoumarin. Interestingly, both fir needle oil and delta-3-carene caused a significant increase in metabolic degradation of 7-ethoxycoumarin, perhaps indicating they upregulate oxidative metabolic processes. Such an action would explain why fir needle oil enhances knockdown, but not 24-h mortality for most of the insecticides studied here, whereas increased bioactivation would explain the synergism of pirimiphos-methyl toxicity., (Published by Oxford University Press on behalf of Entomological Society of America 2023.)

Established dogma concerning the action of insecticidal arthropod-derived peptides (e.g., scorpion toxins), was that they acted on the peripheral nervous system and were excluded from the central nervous system (CNS) by barrier systems. Initial evidence for a CNS-directed toxicological effect following parenteral administration was for a novel peptide from the Hobo spider, Tegeneria agrestis. This toxin was inactive on peripheral sensory and motor nerves, but had a potent excitatory effect on the CNS of larval Musca domestica. Recently, a commercialized formulation of GS-omega/kappa-Hxtx-Hv1a (HXTX), derived from the venom of the Australian blue mountain funnel web spider (Hadronyche versuta) was introduced for use in agriculture by Vestaron Corp. Its primary mode of action was found to be central neuroexcitation via positive allosteric modulation of nicotinic acetylcholine receptors (nAchR) of cockroach neurons. In the present study, this peptide showed hyperexcitation followed by a decrease in firing of the Drosophila melanogaster larval CNS that was prevented by co-exposure to 100 nM α-bungarotoxin (α-BGTX), a classical nAchR noncompetitive antagonist. This effect was mirrored in isobologram analysis, which showed clear antagonism between the two toxins when injected into adult houseflies. Interestingly, U1-agatoxin-Ta1b-QA derived from Tegeneria agrestis (VST-7304) had a similar biphasic action, but showed increased nerve discharge when co-exposed with 100 nM α-BGTX, and had additive effects when injected together with α-BGTX in isobologram analyses. Binary mixtures of HXTX or VST-7304 with 30 nM nicotine showed clear evidence of synergized nerve block, which was also observed for mixtures of HXTX and VST-7304. Taken together, these data suggest that HXTX and VST-7304 have somewhat different and complementary modes of action., (Copyright © 2023 Elsevier Inc. All rights reserved.)

We explored the potential of two sodium channel activators, veratrine and aconitine, as both insecticides and synergists of natural pyrethrins (NP) on Aedes aegypti adults and larvae. Aconitine was more toxic than veratrine, with an LD 50 of 157 ng/mg compared to 376 ng/mg, on the pyrethroid-susceptible Orlando strain, but only aconitine showed significant resistance in the pyrethroid-resistant Puerto Rico strain (RR = 14.6 in topical application and 8.8 in larval bioassay). When applied in mixtures with piperonyl butoxide (PBO) and NP, large synergism values were obtained on the Orlando strain. Aconitine + PBO mixture synergized NP 21.8-fold via topical adult application and 10.2-fold in larval bioassays, whereas veratrine + PBO synergized NP 5.3-fold via topical application and 30.5-fold in larval bioassays. Less synergism of NP was observed on the resistant Puerto Rico strain, with acontine + PBO synergizing NP only 4.1-fold in topical application (8-fold in larval bioassays) and veratrine + PBO synergizing NP 9.5-fold in topical application (13.3-fold in larval bioassays). When alkaloids were applied directly to the mosquito larval nervous system, veratrine was nearly equipotent on both strains, while aconitine was less active on pyrethroid-resistant nerve preparations (no block at 10 μM compared to block at 1 μM on the susceptible strain). The nerve blocking effect of NP was significantly synergized by both compounds on the pyrethroid-susceptible strain by about 10-fold, however only veratrine synergized NP block on the pyrethroid-resistant strain, also showing 10-fold synergism). These results highlight the potential of site II sodium channel activators as insecticides and their ability to synergize pyrethroids, which may extend the commercial lifetime of these chemistries so essential to public health vector control., (Published by Elsevier Inc.)

Nootkatone, a sesquiterpenoid isolated from Alaskan yellow cedar (Cupressus nootkatensis), is known to possess insect repellent and acaricidal properties and has recently been registered for commercial use by the Environmental Protection Agency. Previous studies failed to elucidate the mechanism of action of nootkatone, but we found a molecular overlay of picrotoxinin and nootkatone indicated a high degree of structural and electrostatic similarity. We therefore tested the hypothesis that nootkatone was a GABA-gated chloride channel antagonist, similar to picrotoxinin. The KD 50 and LD 50 of nootkatone on the insecticide-susceptible strain of Drosophila melanogaster (CSOR) showed resistance ratios of 8 and 11, respectively, compared to the cyclodiene-resistant strain of RDL1675, indicating significant cross-resistance. Nootkatone reversed GABA-mediated block of the larval CSOR central nervous system; nerve firing of 78 ± 17% of baseline in the CSOR strain was significantly different from 24 ± 11% of baseline firing in the RDL1675 strain (p = 0.035). This finding indicated that the resistance was expressed within the nervous system. Patch clamp recordings on D. melanogaster central neurons mirrored extracellular recordings where nootkatone inhibited GABA-stimulated currents by 44 ± 9% at 100 μM, whereas chloride current was inhibited 4.5-fold less at 100 μM in RDL1675. Taken together, these data suggest nootkatone toxicity in D. melanogaster is mediated through GABA receptor antagonism., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The risk of honey bee (Apis mellifera L.) exposure to pesticide residues while foraging for nectar and pollen is commonly explored in the context of agroecosystems. However, pesticides are also used in urban and suburban areas for vegetation management, vector control, and the management of ornamental plants in public and private landscapes. The extent to which pesticides pose a health risk to honey bees in these settings remains unclear. We addressed this at a landscape scale by conducting pesticide residue screening analyses on 768 nectar and 862 pollen samples collected monthly over 2 years from honey bee colonies located in urban and suburban areas in eight medium to large cities in California, Florida, Michigan, and Texas (USA). A risk assessment was performed using the US Environmental Protection Agency's BeeREX model whenever an oral toxicity value was available for a compound. Chemical analyses detected 17 pesticides in nectar and 60 in pollen samples during the survey. Approximately 73% of all samples contained no detectable pesticide residues. Although the number of detections varied among the sampled regions, fewer pesticides were detected in nectar than in pollen. Per BeeREX, four insecticides showed a potential acute risk to honey bees: imidacloprid, chlorpyrifos, and esfenvalerate in nectar, and deltamethrin in nectar and pollen. In general, exposure of honey bees to pesticides via nectar and pollen collection was low in urban and suburban areas across the United States, and no seasonal or spatial trends were evident. Our data suggest that honey bees are exposed to fewer pesticides in developed areas than in agricultural ones. Environ Toxicol Chem 2022;41:991-1003. © 2022 SETAC., (© 2022 SETAC.)

To discover new natural materials for insect management, commercially available stored sheep wool was investigated for attractancy to female adult Aedes aegypti mosquitoes. The volatiles from sheep wool were collected by various techniques of headspace (HS) extractions and hydrodistillation. These extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detector (GC-FID) coupled with GC-MS. Fifty-two volatile compounds were detected, many of them known for their mosquito attractant activity. Seven compounds were not previously reported in sheep products. The volatile composition of the extracts varied significantly across collections, depending on the extraction techniques or types of fibers applied. Two types of bioassay were conducted to study attractancy of the sheep wool volatiles to mosquitoes: laboratory bioassays using glass tubes, and semi-field bioassays using large, screened outdoor cages. In bioassays with glass tubes, the sheep wool hydrodistillate and its main component, thialdine, did not show any significant attractant activity against female adult Ae. aegypti mosquitoes. Semi-field bioassays in two large screened outdoor cages, each equipped with a U.S. Centers for Disease Control (CDC) trap and the various bait setups with Vortex apparatus, revealed that vibrating wool improved mosquito catches compared to the setups without wool or with wool but not vibrating. Sheep wool, when vibrated, may release intensively volatile compounds, which could serve as olfactory cues, and play significant role in making the bait attractive to mosquitoes. Sheep wool is a readily available, affordable, and environment-friendly material. It should have the potential to be used as a mosquito management and surveillance component in dynamic bait setups.

Background: The CDC Bottle Bioassay serves as an inexpensive and effective way to screen field-caught mosquitoes against a wide variety of insecticidal active ingredients and commercial formulations, with the goal of detecting resistant individuals. For this study, we assessed how sucrose-water (10% w/v) feeding status impacted the response of Aedes aegypti mosquitoes to select insecticides., Results: Starvation for 24 or 48 h decreased permethrin and malathion mean survival time by about 40%, with little difference in the two starvation times. Similar findings were also observed in a pyrethroid-resistant Puerto Rico strain challenged with permethrin, but these effects were less pronounced. To test the impact of mosquito weight, we measured weight under different 48-h nutritional conditions and found that sugar-water-fed and sugar-only-fed individuals were approximately the same weight (ANOVA, Bonferroni post-test P value >0.999) and that individuals fed water only were considerably lighter than either sugar-water-fed (ANOVA, Bonferroni post-test P value = 0.034) or sugar-only-fed individuals (ANOVA, Bonferroni post-test P value = 0.027) but equal in weight to starved mosquitoes (ANOVA, Bonferroni post-test P value >0.99). Of the nutritional challenges, water-only-fed mosquitoes were the most insecticide tolerant (ANOVA, Bonferroni post-test P values to all other treatments <0.01)., Conclusions: The results indicate insect nutritional status is an important experimental variable, particularly the hydration status of mosquitoes shortly before insecticide exposure. Moreover, as significant differences were observed between starved and component-fed (water, sugar, or sugar and water) mosquitoes in a pyrethroid-resistant strain, some caution is appropriate when interpreting resistance/susceptibility diagnoses with this bioassay. © 2021 Society of Chemical Industry. This article has been contributed to by US Government employees and their work is in the public domain in the USA., (© 2021 Society of Chemical Industry. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Background: Varroa destructor is among the greatest threats to honey bee health worldwide. Acaricides used to control Varroa are becoming increasingly ineffective due to resistance issues, prompting the need for new compounds that can be used for control purposes. Ideally, such compounds would exhibit high toxicity to Varroa while maintaining relatively low toxicity to bees and beekeepers. We characterized the lethal concentrations (LC 50 ) of amitraz, matrine, FlyNap®, the experimental carbamates 2-((2-ethylbutyl)thio)phenyl methylcarbamate (1) and 2-(2-ethylbutoxy)phenyl methylcarbamate (2), and dimethoate (positive control) for Varroa using a glass vial assay. The test compounds also were applied to honey bees using an acute contact toxicity assay to determine the adult bee LD 50 for each compound., Results: Amitraz was the most toxic compound to Varroa, but carbamate 2 was nearly as active (within 2-fold) and the most selective due to its lower bee toxicity, demonstrating its promise as a Varroa control. While carbamate 1 was less toxic to honey bees than was amitraz, it was also 4.7-fold less toxic to the mites. Both matrine and FlyNap® were relatively ineffective at killing Varroa and were moderately toxic to honey bees., Conclusion: Additional testing is required to determine if carbamate 2 can be used as an effective Varroa control. As new chemical treatments are identified, it will be necessary to determine how they can be utilized best alongside other control techniques as part of an integrated pest management program. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Previous studies have shown that insect cell cultures stop dividing, form clumps, and can be induced to grow processes reminiscent of axons, when the culture medium is supplemented with 20-hydroxyecdysone, insulin, or an agent that mimics their action, such as the ecdysone agonist, methoxyfenozide. Those cell growing processes resemble nerve cells, and the present study evaluates the ultrastructure of these cultures by transmission electron microscopy. Sf 21 cells treated with 20-hydroxyecdysone (with or without veratridine amendment) and subjected to ultrastructural analysis had a similar somatic appearance to control cells, with slight changes in organelles and organization, such as a greater number of cytoplasmic vacuoles and mitochondrial granules. Finger-like projections were observed between control and treated cells. However, no structural markers of synaptic contacts (e.g., vesicles or synaptic thickenings) were observed in controls, 20-hydroxyecdysone, or 20-hydroxyecdysone + veratridine treated cells. It is concluded that additional agents would be required to induce functional synaptogenesis in Sf 21 cells.

It is important to identify repellents that can provide reliable protection from arthropod biting and prevent arthropod-borne diseases, such as malaria. In the present study, the spatial repellent activity and toxicity of two novel pyridinyl amides ( 1 and 2 ) were evaluated against Anopheles albimanus , Anopheles quadrimaculatus , and Anopheles gambiae . In vapor repellency bioassays, compound 2 was generally more effective than DEET and 2-undecanone, while compound 1 was about as active as these standards. Overall, transfluthrin was the most active compound for inducing anopheline mosquito repellency, knockdown, and lethality. Although they were not the most active repellents, the two experimental amides produced the largest electroantennographic responses in female antennae. They also displayed modest toxicity to anopheline mosquitoes. Significant synergism of repellency was observed for the mixture of a pyrethroid-derived acid and the repellent 2-undecanone against anopheline mosquitoes, similar to that observed previously in Aedes aegypti . Overall, this study provides insight for further synthesis of alternative amide compounds for use as spatial treatments., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Resistance to currently utilized chemical insecticidal agents represents a significant threat to public health and food security worldwide. Better understanding the neurophysiological effects of available and candidate insecticidal molecules is valuable for characterizing the mechanisms of insecticide resistance, as well as the design and study of novel control chemistries. In this paper, we describe a method of recording nerve firing from the central nervous system of Aedes aegypti fourth instar larvae. In short, mosquito larvae were immobilized by placing small pins through the head and siphon of the larvae in a wax dish, ventral side down. A single, longitudinal, dorsal incision from the distal abdomen to the pronotum of the larva was made, the alimentary canal removed, and the ventral nerve cord severed between the second and third abdominal ganglia. A recording suction electrode was connected directly to axons within the severed end of the connective in a novel way to record nerve firing in the ventral nerve cord at a high signal-to-noise ratio with conventional electrophysiological equipment. Using this novel method, we report the effects of four neuroactive compounds using this method: octopamine, pilocarpine, nicotine, and γ-aminobutyric acid (GABA). The utility of this recording technique for elucidating target site mechanisms involved in insecticide resistance is demonstrated with p,p'-dichlorodiphenyltrichlorethane (DDT) and its difluoro analog (DFDT)., (Published by Elsevier Ltd.)

Liriodenine is a biologically active plant alkaloid with multiple effects on mammals, fungi, and bacteria, but has never been evaluated for insecticidal activity. Accordingly, liriodenine was applied topically in ethanolic solutions to adult female Anopheles gambiae, and found to be mildly toxic. Its lethality was synergized in mixtures with dimethyl sulfoxide and piperonyl butoxide. Recordings from the ventral nerve cord of larval Drosophila melanogaster showed that liriodenine was neuroexcitatory and reversed the inhibitory effect of 1 mM GABA at effective concentrations of 20-30 μM. GABA antagonism on the larval nervous system was equally expressed on both susceptible and cyclodiene-resistant rdl preparations. Acutely isolated neurons from Periplaneta americana were studied under patch clamp and inhibition of GABA-induced currents with an IC 50 value of about 1 μM were observed. In contrast, bicuculline did not reverse the effects of GABA on cockroach neurons, as expected. In silico molecular models suggested reasonable structural concordance of liriodenine and bicuculline and isosteric hydrogen bond acceptor sites. This study is the first assessing of the toxicology of liriodenine on insects and implicates the GABA receptor as one likely neuronal target, where liriodenine might be considered an active chemical analog of bicuculline., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Previous studies have shown that blockers of voltage-gated potassium (Kv) channels, such as 4-aminopyridine (4-AP) and 2-methoxy-N-((1-phenylcyclopentyl)methyl)benzamide (2-MPB) synergized pyrethroid toxicity as well, or better than, piperonyl butoxide. The present study assessed the involvement of different Kv channels as possible pyrethroid synergist targets in Drosophila melanogaster. Three Kv1 mutants (Sh 5 , Sh 133 , and Sh M ) and one Kv2 mutant (Shab 3 ) were tested. All Kv1 mutant flies showed increased sensitivity to permethrin in topical and glass contact toxicity assays, of 2- to 11-fold. Central nervous system (CNS) recordings of larval D. melanogaster showed a similar pattern of increased sensitivity. Potentiated effects were also observed with deltamethrin on the mutants Sh 5 (30- to 35-fold) and Sh 133 (33- to 47-fold), but the mutant Sh M showed little change in sensitivity. In contrast, the Shab 3 strain showed toxicity and physiological effects of both pyrethroids that were similar to the susceptible OR strain. Thus, some K + channel mutations mimicked the synergistic effect of channel blockers. Additional studies showed that Shab 3 had the highest sensitivity to 4-AP in topical assays, and the Shaker-null mutants, Sh M and Sh 133 showed greater sensitivity to 2-MPB in CNS recordings of larval D. melanogaster. These results suggest that Kv1 channels are a useful synergist target for pyrethroids, as assessed both in whole insects and at the level of the nervous system. Thus, Kv1-targeting compounds can potentially serve as insect control tools to reduce pyrethroid use via synergistic action., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The use of N-aryl amide derivatives as spatially acting insecticides remains relatively unexplored. To expand this knowledge, we synthesized eighty-nine N-aryl amide analogues and screened them for mortality against an insecticide-susceptible strain of Aedes aegypti mosquitoes, Orlando (OR), using a vapor exposure glass tube assay. Of the screened compounds, twenty-two produced >92% mortality at 24 h and warranted further investigation to determine LC 50 values. Fifteen of these analogues had LC 50 values within 2 orders of magnitude of transfluthrin, and of significant interest, N -(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (compound 70 ) was nearly as potent as transfluthrin and exhibited greater toxicity than metofluthrin when screened against OR A. aegypti . Compounds exhibiting potent toxicity against OR A. aegypti or whose structure-activity relationship potentially offered beneficial insights into structure optimization were screened against the insecticide-resistant, Puerto Rico (PR), strain of A. Aegypti, and it was discovered that not only did these N-arylamides typically show little resistance, some such as N -(2,6-dichloropyridin-4-yl)-2,2,3,3,4,4,4-heptafluorobutanamide (compound 36 ) and 2,2,3,3,4,4,4-heptafluoro- N -(3,4,5-trifluorophenyl)butanamide (compound 40 ) were actually more potent against the PR mosquitoes. Due to this promising insecticidal activity, five compounds were administered orally to mice to determine acute oral rodent toxicity. All five compounds were found to have mouse oral toxicity LD 50 values well above the minimum safe level as set by the Innovative Vector Control Consortium (50 mg/kg). In addition to the promising biological activity documented here, we report the structure-activity relationship analysis used to guide the derivatization approach taken and to further inform future efforts in the development of N-arylamides as potential resistance-breaking, spatially acting insecticides.

Background: The compound 2-((4-ethyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)thio)-N-(4-ethylphenyl) acetamide (VUAA1) is reported to be an odorant receptor co-receptor (Orco) agonist in insects with potential use as an insect repellent. For this study, the biological activity of VUAA1 was investigated in several bioassays with Aedes aegypti, including adult contact, spatial repellency, and larval repellency assays, as well as topical, injection, and feeding toxicity assays. Neurophysiological action was further explored by analysis of fruit fly central nervous system firing, cockroach axon recordings, patch clamp analysis of Kv2 potassium channel, and acetylcholinesterase inhibition studies. Finally, the metabolic impact on the toxicity of VUAA1 was explored by applying it in combination with established metabolic synergists., Results: In repellency and bite protection screens, VUAA1 showed little activity against adult mosquitoes, apparently due to its low volatility, since its effectiveness was increased by heating or mixing with transfluthrin acid and citronella oil. It did produce measurable repellency of mosquito larvae that was more potent than N,N-diethyl-m-toluamide (DEET). Overall, VUAA1 showed low acute toxicity to both insects and mice, and it was weakly synergized by triphenyl phosphate. There was no observed cross-resistance in a pyrethroid-resistant strain of Anopheles gambiae. VUAA1 showed a two-phase effect on the central nervous system, with neuroexcitation at 1 μmol L -1 and an inhibitory effect at 100 μmol L -1 that may relate to block of Kv2 potassium channels., Conclusions: VUAA1 presented low toxicity, similar to other insect repellents. Its limited solubility, low volatility, and resulting poor adult repellency without additional adjuvants may restrict the utility of VUAA1 in typical public health applications. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Background: The purpose of this research was to explore the possibility of co-applying pyrethroids (agonists of voltage-sensitive sodium channels) with potassium channel blockers in order to potentiate the neurological effects of pyrethroids on Anopheles gambiae. We hypothesized that the toxicity of pyrethroids caused by persistent sodium currents would be augmented by blockage of outward potassium current flow, which normally repolarizes the membrane potential during a nerve membrane action potential., Results: Topical treatments with LD 10 s (10% mortality doses) of synergists were given with pyrethroids. 2S-65465 (2S) showed the best synergism of permethrin (8.6-fold) and deltamethrin (7.2-fold), whereas piperonyl butoxide and 4-aminopyridine only showed 2.2- to 3.4-fold synergism with these pyrethroids. In electrophysiological recordings of Periplaneta americana giant axons, 2S (10 μm) and 4-AP (30 μm) caused multiple spikes after a single stimulation. Permethrin at 10 μm showed significant summating depolarization (4.5 ± 1.1 mV) after a train of ten stimuli were applied at 5 Hz, and deltamethrin at 0.03 μm showed significant membrane depolarization of 2.9 ± 0.4 mV without stimuli. 2S at 0.3 μm and 4-AP at 1-3 μm significantly synergized the effects of 3 μm permethrin and 0.01 μm deltamethrin., Conclusions: Co-application of potassium channel blockers 2S and 4-AP with pyrethroids can synergize the mosquitocidal activities on An. gambiae, and these activities are correlated with synergistic effects at the level of the nerve membrane. If deployed in the field, this approach can potentially reduce the amount of chemicals needed for effective control of mosquitoes. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Delivery of compounds to the brain is critical for the development of effective treatment therapies of multiple central nervous system diseases. Recently a novel insect-based brain uptake model was published utilizing a locust brain ex vivo system. The goal of our study was to develop a priori, in silico cheminformatic models to describe brain uptake in this insect model, as well as evaluate the predictive ability. The machine learning program Orange® was used to evaluate several machine learning (ML) models on a published data set of 25 known drugs, with in vitro data generated by a single laboratory group to reduce inherent inter-laboratory variability. The ML models included in this study were linear regression (LR), support vector machines (SVN), k-nearest neighbor (kNN) and neural nets (NN). The quantitative structure-property relationship models were able to correlate experimental logCtot (concentration of compound in brain) and predicted brain uptake of r 2  > 0.5, with the descriptors log(P*MW -0.5 ) and hydrogen bond donor used in LR, SVN and KNN, while log(P*MW -0.5 ) and total polar surface area (TPSA) descriptors used in the NN models. Our results indicate that the locust insect model is amenable to data mining chemoinformatics and in silico model development in CNS drug discovery pipelines., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The combined effect of global warming and insecticide exposure on the spread of mosquito-borne diseases is poorly studied. In our opinion, more resources should be diverted to this topic to further research efforts and deal with this increasing threat. It is particularly important to determine how Aedes, Anopheles, and Culex vector species cope with insecticide exposure under warming temperatures, as well as how both stressors may impact the activity of mosquito biocontrol agents. Herein, we promote a discussion on the topic, fostering a research agenda with insights for the longer-term implementation of mosquito control strategies under the Integrated Vector Management framework., Competing Interests: Declaration of competing interest The authors declare no conflict of interests about the publication of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

With insecticide-resistant mosquito populations becoming an ever-growing concern, new vector control technologies are needed. With the lack of new chemical classes of insecticides to control mosquito populations, the development of novel synergists may improve the performance of available insecticides. We screened a set of 20 plant essential oils alone and in combination with natural pyrethrins against Aedes aegypti (Orlando) female adult mosquitoes to assess their ability to synergize this natural insecticide. A co-toxicity factor analysis was used to identify whether plant oils modulated the toxicity of natural pyrethrins antagonistically, additively, or synergistically. Both knockdown at 1 h and mortality at 24 h were monitored. A majority of oils increased the toxicity of natural pyrethrins, either via an additive or synergistic profile. Many oils produced synergism at 2 µg/insect, whereas others were synergistic only at the higher dose of 10 µg/insect. Amyris, cardamom, cedarwood, and nutmeg East Indies (E.I.) oils were the most active oils for increasing the mortality of natural pyrethrins at 24 h with co-toxicity factors greater than 50 at either or both doses. A number of oils also synergized the 1 h knockdown of natural pyrethrins. Of these, fir needle oil and cypress oils were the most successful at improving the speed-of-action of natural pyrethrins at both doses, with co-toxicity factors of 130 and 62, respectively. To further assess the co-toxicity factor method, we applied selected plant essential oils with variable doses of natural pyrethrins to calculate synergism ratios. Only the oils that produced synergistic co-toxicity factors produced statistically significant synergism ratios. This analysis demonstrated that the degree of co-toxicity factor correlated well with the degree of synergism ratio observed (Pearson correlation coefficient r = 0.94 at 2 µg/insect; r = 0.64 at 10 µg/insect) and that the co-toxicity factor is a useful tool in screening for synergistic activity.

The productivity and survival of managed honey bee colonies is negatively impacted by a diverse array of interacting factors, including exposure to agrochemicals, such as pesticides. This study investigated the use of volatile heterocyclic amine (HCA) compounds as potential short-term repellents that could be employed as feeding deterrents to reduce the exposure of bees to pesticide-treated plants. Parent and substituted HCAs were screened for efficacy relative to the repellent N , N -diethyl-meta-toluamide (DEET) in laboratory and field experiments. Additionally, electroantennogram (EAG) recordings were conducted to determine the level of antennal response in bees. In video-tracking recordings, bees were observed to spend significantly less time with an HCA-treated food source than an untreated source. In a high-tunnel experiment, the HCA piperidine was incorporated in a feeding station and found to significantly reduce bee visitations relative to an untreated feeder. In field experiments, bee visitations were significantly reduced on melon flowers ( Cucumis melo L.) and flowering knapweed ( Centaurea stoebe L.) that were sprayed with a piperidine solution, relative to untreated plants. In EAG recordings, the HCAs elicited antennal responses that were significantly different from control or vehicle responses. Overall, this study provides evidence that HCAs can deter individual bees from food sources and suggests that this deterrence is the result of antennal olfactory detection. These findings warrant further study into structure-activity relationships that could lead to the development of short-term repellent compounds that are effective deterrents to reduce the contact of bees to pesticide-treated plants.

Research on the functions of insect chemoreceptors have primarily focused on antennae (olfactory receptors) and mouthparts (gustatory receptors). However, chemoreceptive sensilla are also present on other appendages, such as the leg tarsi and the anterior wing margin, and their specific roles in chemoreception and mosquito behavior remain largely unknown. In this study, electrophysiological analyses in an electroantennogram recording format were performed on Aedes aegypti (L., Diptera: Culicidae) antennae, mouthparts, tarsi, and wings during exposure to a variety of insect repellent and attractant compounds. The results provide evidence that the tarsi and wings can sense chemicals in a gaseous form, and that the odors produce differing responses on different appendages. The most consistent and strongest response occurred when exposed to triethylamine (TEA). Antennae and mouthparts showed nearly identical responses pattern to all tested compounds, and their rank orders of effectiveness were similar to those of fore- and mid-leg tarsi. Hindleg tarsi only responded to TEA, indicating that the hind legs are not as chemoreceptive. Wings responded to a range of odorants, but with a different rank order and voltage amplitude. Insights gleaned into the function of these appendages in insect chemoreception are discussed., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A total of 115 aryl amides were synthesized and screened for vapor repellency against the Orlando (OR) strain of Aedes aegypti mosquitoes. Of these compounds, 29 had 1 h repellency EC 50 values comparable to or better than N , N -diethyl- meta -toluamide (DEET, 1 h EC 50 value of 35 μg/cm 2 ), with 2,2,3,3,3-pentafluoro- N -(4-fluorophenyl)propenamide ( 53 ) and 2,2,3,3,4,4,4-heptafluoro- N -(3,4,5-trifluorophenyl)butanamide ( 101 ) exhibiting the most potent EC 50 values of 4.5 and 2.9 μg/cm 2 , respectively. The cross-resistance of select, highly potent, derivatives against the pyrethroid-resistant Puerto Rico (PR) strain of A. aegypti was also investigated, and little to no resistance was observed. When synergized with 1 R - trans -permethrinic acid (TFA), compound 101 had a 1 h EC 50 value 6 times lower than metofluthrin against OR and 40 times lower against PR mosquitoes. Additionally, preliminary mammalian oral toxicity was screened for compounds 69 and 101 , and both exhibited LD 50 values of >2000 mg/kg. The structure-activity relationship analysis, which guided the synthesis of these derivatives, is given, and key trends are highlighted to inform future analogue design.

New insecticides are urgently needed for the control of arthropod vectors of public health diseases. As resistance to many insecticides used for the control of public health pests is ubiquitous, all available chemistries should be evaluated for their potential to effectively control both insecticide-susceptible and insecticide-resistant strains of mosquitoes. This study aimed to evaluate p-p'-difluoro-diphenyl-trichloroethane (DFDT) as a mosquito control technology and relate its activity to that of DDT. We found that topical DFDT was significantly less toxic than DDT to both pyrethroid-susceptible and pyrethroid-resistant strains of Anopheles gambiae and Aedes aegypti. Direct nervous system recording from Drosophila melanogaster CNS demonstrated that DFDT is approximately 10-times less potent than DDT at blocking nerve firing, which may explain its relatively lower toxicity. DFDT was shown to be at least 4500 times more vapor-active than DDT, with an LC 50 in a vapor toxicity screening assay of 2.2 μg/cm 2 . Resistance to DFDT was assessed in two mosquito strains that possess target-site mutations in the voltage-gated sodium channel and upregulated metabolic activity. Resistance ratios for Akdr (An. gambiae) and Puerto Rico (Ae. aegypti) strains were 9.2 and 12.2, respectively. Overall, this study demonstrates that DFDT is unlikely to be a viable public health vector control insecticide., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aedes aegypti L. (Diptera: Culicidae) is one of the most medically important mosquito species, due to its ability to spread viruses of yellow fever, dengue fever, and Zika in humans. In this study, the insecticidal activity of 17 plant essential oils was evaluated via topical application against two strains of Ae. aegypti mosquito, Orlando (insecticide-susceptible) and Puerto Rico (pyrethroid-resistant). Initial screens with the Orlando strain showed that cucumber seed oil (2017 sample) was the most toxic, followed by sandalwood and thyme oil. When the essential oils were mixed with permethrin, they failed to show any significant synergism of insecticidal activity. Sandalwood and thyme oils displayed consistently high mortality against the resistant Puerto Rico strain, with low resistance ratios of 2.1 and 1.4, respectively. In contrast, cucumber seed oil showed significantly less activity against Puerto Rico mosquitoes, with a resistance ratio of 45. Bioactivity-guided fractionation of the 2017 sample of cucumber seed oil sample via flash column chromatography produced 11 fractions, and gas-chromatography/mass spectrometry analysis revealed that the three active fractions were contaminated with 0.33, 0.36, and 0.33% of chlorpyrifos-methyl, an organophosphorus insecticide, whereas inactive fractions did not show any trace of it. These results suggested that the insecticidal activity of cucumber seed oil was probably due to the presence of the insecticide, later confirmed with a clean batch of cucumber seed oil obtained in 2018, which showed negligible insecticidal activity. These findings demonstrate clearly the need for essential oil analysis to confirm purity before any claims are made about pesticidal potency., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite + ® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite + ® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite + ® efficacy has been. reduced or lost for Varroa mites., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Pyrethroids are one of the most commonly used classes of insecticides, and their acid and alcohol components are esterase degradation products, usually considered to be biologically inactive. In this study, it was found that several pyrethroid acids had a spatial repellent activity that was greater than DEET, often more active than the parent pyrethroids, and showed little cross resistance in a pyrethroid-resistant Puerto Rico strain of Aedes aegypti mosquitoes. Further investigation revealed that the acids can synergize not only contact repellent standards but also other pyrethroid components as well as the parent pyrethroids themselves. Synergism by the pyrethroid acids is expressed as both increased spatial repellency and vapor toxicity as well as human bite protection. Electrophysiological studies confirmed that pyrethroid acids (100 μM) had no effect on neuronal discharge in larval Drosophila melanogaster CNS and were detected by electroantennography, and there was little resistance to olfactory sensing of these acids in antennae from Puerto Rico strain mosquitoes carrying kdr mutations. Thus, the data suggest that the pyrethroid acids have a different mode of action than the parent pyrethroids, unrelated to the voltage-sensitive sodium channel. The results highlight the potential of pyrethroid acids to be useful in future repellent formulations.

Multiple interacting stressors negatively affect the survival and productivity of managed honey bee colonies. Pesticides remain a primary concern for beekeepers, as even sublethal exposures can reduce bee immunocompetence, impair navigation, and reduce social communication. Pollinator protection focuses on pesticide application guidelines; however, a more active protection strategy is needed. One possible approach is the use of feeding deterrents that can be delivered as an additive during pesticide application. The goal of this study was to validate a laboratory assay designed to rapidly screen compounds for behavioral changes related to feeding or feeding deterrence. The results of this investigation demonstrated that the synthetic Nasonov pheromone and its terpenoid constituents citral, nerol, and geraniol could alter feeding behavior in a laboratory assay. Additionally, electroantennogram assays revealed that these terpenoids elicited some response in the antennae; however, only a synthetic Nasonov pheromone, citral, and geraniol elicited responses that differed significantly from control and vehicle detections., Competing Interests: The authors declare no conflict of interest.

Background: The mosquito, Aedes aegypti (Diptera: Culicidae), is a vector of dengue fever, zika, chikungunya, and yellow fever viruses, and in many areas possesses significant levels of resistance to pyrethroids. Behavioral performance was assessed in 15, 30, and 60 min exposures in a high throughput vapor phase spatial repellency assay to three contact repellent standards: N,N-diethyl-3-methylbenzamide (DEET), ethyl 3-[acetyl(butyl)amino] propanoate (IR3535), and 2-undecanone, as well as pyrethrum extract, transfluthrin, and metofluthrin in susceptible (Orlando) and a pyrethroid-resistant Puerto Rico strain of Aedes aegypti. Additionally, electroantennographic studies were used to investigate the antennal sensitivities to these compounds in both strains., Results: Resistance was found to all tested insect repellents in the Puerto Rico strain of Ae. aegypti. Resistance ratios at the different time points were about 2 for DEET, 3 for 2-undecanone, and 12 for IR3535. Resistance was also observed to pyrethrum extract (∼9-fold), transfluthrin (∼5-fold), and metofluthrin (∼48-fold) in repellent behavioral response. Electrophysiological analysis found decreased antennal sensitivity to all repellents tested, consistent with their behavioral effects., Conclusion: The reduced sensitivity to these repellents may represent a fitness cost arising from the kdr mutation present in Puerto Rico Aedes aegypti. This work highlights the need for understanding collateral effects from the evolution of pesticide resistance in mosquitoes, and the importance of finding alternative strategies to control resistance development. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The adulticidal, larvicidal, and repellent activity of 18 trifluoromethylphenyl amides (TFMPAs) was determined against Aedes aegypti mosquitoes. The compounds studied are the third generation designed from active structures of the previous two generations. N-(3,5-Bis(trifluoromethyl)phenyl)-2-chloroacetamide (8f) and N-(3,5-bis(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (8h) were most active against 1st stage Ae. aegypti larvae with LC 50 values of 125 and 2.53 μM; for comparative purposes, the published LC 50 for fipronil is 0.014 μM. Compound 8h was the most toxic against adult female Ae. aegypti with an LD 50  = 2.12 nmol/mg, followed by 8f, and N-(3,5-bis(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide (8g) with LD 50 values of 4.27 and 4.73 nmol/mg, respectively, although these compounds were significantly less toxic than fipronil against adult female Ae. aegypti. Compounds N-(2-(trifluoromethyl)phenyl)butyramide (9c), N-(2-(trifluoromethyl)phenyl)pentanamide (9d) and N-(2-(trifluoromethyl)phenyl)hex-5-enamide (9e) were the best repellents for female Ae. aegypti, with minimum effective dosages (MEDs) of 0.026, 0.052, and 0.091 μmol/cm 2 , respectively, compared to DEET at 0.052 μmol/cm 2 . Out of 52 TFMPAs (total number of compounds from three generations of this research) compound 9c was the most active repellent along with two synthesized in our previous studies, 2-chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) and 2,2,2-trifluoro-N-(2-(trifluoromethyl)phenyl)acetamide (4c)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Spatial repellents are an essential tool for personal protection against mosquitoes that bite and transmit disease pathogens to humans. Current repellent screening methods, such as olfactometers and alternative choice tests, are complex systems that require a relatively large quantity of compound (mg). The present study validates a high-throughput spatial repellent screening method using a glass tube that has the ends covered with netting, in addition to treated filters and plastic end caps. The apparatus occupies relatively little space, is easy to decontaminate, and requires small amounts of compound (μg). In a horizontal tube orientation, DEET (N,N-diethyl-meta-toluamide), citronella oil and IR3535 had 1 h half repellent concentration (EC 50 ) values of 32, 32 and 298 μg/cm 2 , respectively, against the Orlando strain of Aedes aegypti (L.) (Diptera: Culicidae). Vertical tube orientation increased EC 50 values by approximately two-fold, except IR3535, which remained essentially unchanged. Transfluthrin showed concentration-dependent spatial repellency (1 h EC 50  = 0.5 μg/cm 2 ) without any knockdown, although only in vertical tubes. Transfluthrin showed 50% knockdown in 1 h at 0.5 μg/cm 2 and 50% mortality at 0.15 μg/cm 2 in horizontal tubes. In conclusion, this high-throughput screening method is useful for assessing vapour toxicity and the spatial repellency of candidate molecules prior to semi-field and field studies., (© 2019 The Royal Entomological Society.)

Controversies surrounding the safety of N,N-diethyl-meta-toluamide (DEET) when used as an insect repellent are centered around conflicting findings in the scientific literature and inaccurate reporting in the public media. Lethal cases of DEET poisoning are few, and usually due to deliberate or other overdoses that ignore product label instructions. Deleterious effects of DEET typically involve skin reactions and even when encephalopathies, such as seizures, occur they often abate without sequelae. Recent mode-of-action studies prove it has little direct effect on acetylcholinesterase, and have identified G protein-coupled receptors as a site of action deserving of further investigation. Studies with pregnant women found that DEET had no effect on the developing fetus from proper use and its continued deployment as a repellent is endorsed by both the Centers for Disease Control and Prevention and the Environmental Protection Agency, with specific recommendations of how it should be used on children. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Spatial repellents can reduce fecundity and interrupt oviposition behavior in Aedes aegypti. Yet, it is unclear if short exposure times, resistant phenotypes, and other aspects of spatial repellents can impact these effects on mosquito reproduction. To address these issues, pyrethroid susceptible, pyrethroid resistant, and field strains of Ae. aegypti were used to evaluate the extent to which fecundity and oviposition behavior are affected following metofluthrin exposure. Mosquitoes were exposed for 60 s to a sub-lethal dose (LC30) of metofluthrin before blood feeding and allowed 72 h to become gravid before evaluation in an oviposition bioassay for an additional 72 h. Metofluthrin-exposed susceptible, field, and to a lesser extent resistant strain Ae. aegypti showed oviposition across fewer containers, less egg yield, less egg viability, and reduced larval survivorship in hatched eggs compared to unexposed cohorts. Susceptible mosquitoes retained some eggs at dissection following bioassays, and in one case, melanized eggs retained in the female. Treated resistant and field strain F1 larvae hatched significantly earlier than unexposed cohorts and resulted in increased larval mortality in the first 3 d after oviposition. Upon laying, the treated field strain had incompletely melanized eggs mixed in with viable eggs. The treated field strain also had the lowest survivorship of larvae reared from bioassay eggs. These results indicate that metofluthrin could succeed in reducing mosquito populations via multiple mechanisms besides acute lethality. With the available safety data, pre-existing spatial repellent registration, and possibilities for other outdoor delivery methods, metofluthrin is a strong candidate for transition into broader mosquito abatement operations., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

New insecticides are needed for control of disease-vectoring mosquitoes and this research evaluates the activity of new carbamate acetylcholinesterase (AChE) inhibitors. Biochemical and toxicological characterization of carbamates based on the parent structure of terbam, 3- tert -butylphenyl methylcarbamate, was performed. In vitro enzyme inhibition selectivity ( Anopheles gambiae versus human) was assessed by the Ellman assay, as well as the lethality to whole insects by the World Health Organization (WHO) paper contact assay. Bromination at the phenyl C6 position increased inhibitory potency to both AChEs, whereas a 6-iodo substituent led to loss of potency, and both halogenations caused a significant reduction of mosquitocidal activity. Similarly, installation of a hexyl substituent at C6 drastically reduced inhibition of Ag AChE, but showed a smaller reduction in the inhibition of hAChE. A series of 4-carboxamido analogs of the parent compound gave reduced activity against Ag AChE and generally showed more activity against hAChE than Ag AChE. Replacement of the 3- t -buyl group with CF 3 resulted in poor anticholinesterase activity, but this compound did have measurable mosquitocidal activity. A series of methyl- and fluoro- analogs of 3-trialkylsilyl compounds were also synthesized, but unfortunately resulted in disappointing activity. Finally, a series of sulfenylated proinsecticides showed poor paper contact toxicity, but one of them had topical activity against adult female Anopheles gambiae . Overall, the analogs prepared here contributed to a better understanding of carbamate structure-activity relationships (SAR), but no new significant leads were generated.

Background: The effects of chronic exposure to two neonicotinoids (clothianidin and imidacloprid) and two organophosphates (chlorpyrifos and dimethoate) on survival, developmental rate and larval weight of honey bee larvae reared in vitro were determined. Diets containing chemicals were fed to larvae with the range of concentrations for each compound based on published acute toxicity experiments and residues found in pollen and nectar, both components of the larval diet., Results: Four concentrations of each compound and controls were tested: chlorpyrifos: 0.5, 0.8, 1.2, 8 mg/L; clothianidin: 0.1, 0.4, 2, 10 mg L -1 ; dimethoate: 0.02, 1, 6, 45 mg L -1 ; imidacloprid: 0.4, 2, 4, 10 mg L -1 ; positive control: dimethoate (45 mg L -1 ); solvent control: acetone or methanol; and negative control. A significant decrease in survival, relative to the solvent control, occurred in the 0.8, 1.2 and 8 mg L -1 chlorpyrifos, 0.4, 2 and 10 mg L -1 clothianidin, and 45 mg L -1 dimethoate diets, but not the imidacloprid diets., Conclusion: The treatment of larval diets with clothianidin, dimethoate and imidacloprid did not affect survival, developmental rate, or weight of immature honey bees; however, treatment with chlorpyrifos did. Overall, our results are valuable for evaluating the chronic toxicity of these pesticides to developing honey bees. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Background: Mosquito mortality has been documented in numerous studies of spatial repellents but the concentration-dependent toxicity of spatial repellent vapors has not been documented. To address this issue, prallethrin, flumethrin, metofluthrin, transfluthrin, and meperfluthrin were selected for comparative study against Aedes albopictus (Skuse), Ae. aegypti (L.), Culex quinquefasciatus Say, and Anopheles quadrimaculatus Say. Mosquito were exposed to vapors of each chemical for 2, 4, and 24 h with mortality recorded at each time point. A second experiment involved exposing mosquitoes to vapors for 2 h, then transferring them to untreated holding containers and held for 24 h. For these mosquitoes, readings were only taken after 24 h to allow for metabolic detoxification and recovery., Results: LC 50 and LC 90 data indicated that transfluthrin and meperfluthrin had the greatest toxicity across all species, followed by metofluthrin, prallethrin, and flumethrin., Conclusion: Our findings, through the direct comparison of these compounds, suggest that transfluthrin, meperfluthrin, and metofluthrin be considered for further development. The vapor toxicity for the aforementioned compounds significantly exceeds prallethrin, which is currently market available as an adulticidal active ingredient in public health pest control. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Chlorothalonil is a broad-spectrum fungicide and diflubenzuron is an insect growth regulator used to control many insect larvae feeding on agricultural, forest and ornamental plants. Honey bee larvae may be exposed to both via contaminated pollen, in the form of beebread, added to their diet by their adult nurse sisters. In this study, we determined how single (acute: 72 h mortality) and repeated (chronic: mortality until emergence as adults) exposure to chlorothalonil and diflubenzuron in their diet affected honey bee larvae reared in vitro. The tested doses of chlorothalonil (20, 100, or 200 mg/L) did not impact 72 h larval mortality acutely relative to that of the solvent control. The 72 h mortality of larvae exposed to 1.6 mg/L and higher doses of diflubenzuron acutely in their diet (47.2-63.9% mortality) was significantly higher than that of larvae fed the solvent control, with no predictable dose dependent pattern observed. In the chronic toxicity tests, consuming an artificial diet with 30 or 100 mg/L chlorothalonil and 0.8, 1.3 or 2 mg/L diflubenzuron significantly lowered the survival of honey bee larvae over that of larvae feeding on the solvent control diet. We calculated risk quotients (RQs) for both compounds using the data we generated in our experiments. Collectively, the RQs suggest that neither compound is likely to affect larval mortality directly at field relevant doses given that pollen composes only a fraction of the total larval diet. Nevertheless, our data do not preclude any sublethal effects that chronic exposure to either compound may cause., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Anopheles gambiae Giles (Diptera: Culicidae) is the most prolific malaria vector in sub-Saharan Africa, where widespread insecticide resistance has been reported. An. gambiae laboratory strains are commonly used to study the basic biology of this important mosquito vector, and also in new insecticide discovery programs, where insecticide-susceptible and -resistant strains are often used to screen new molecules for potency and cross-resistance, respectively. This study investigated the toxicity of permethrin, a Type-I pyrethroid insecticide, and etofenprox, a non-ester containing pyrethroid insecticide, against An. gambiae at three life stages. This characterization was performed with susceptible (G3; MRA-112) and resistant (A kdr ; MRA-1280) An. gambiae strains; the A kdr strain is known to contain the L1014F mutation in the voltage-sensitive sodium channel. Surprisingly, etofenprox displays a lower level of resistance than permethrin against all stages of mosquitoes, except in a headless larval paralysis assay designed to minimize penetration factors. In first-instar An. gambiae larvae, permethrin had significant resistance, determined by the resistance ratio (RR 50 = 5), but etofenprox was not significantly different (RR 50 = 3.4) from the wild-type strain. Fourth-instar larvae displayed the highest level of resistance for permethrin (RR 50 = 108) and etofenprox (RR 50 = 35). Permethrin (PC 50 = 2 ppb) and etofenprox (PC 50 = 9 ppb) resulted in headless larval paralysis (5-h), but resistance, albeit lower, was still present for permethrin (RR 50 = 5) and etofenprox (RR 50 = 6.9). In adult female mosquitoes, permethrin displayed higher resistance (RR 50 = 14) compared to etofenprox (RR 50 = 4.3). The level of etofenprox resistance was different from that previously reported for a similar Akron An. gambiae laboratory strain (MRA-913). The chemical synergists piperonyl butoxide (PBO) and diethyl maleate (DEM) were able to synergize permethrin, but not etofenprox in the resistant strain (A kdr ). In conclusion, multiple mechanisms are likely involved in pyrethroid resistance, but resistance profiles are dependent upon selection. Etofenprox is an effective insecticide against An. gambiae in the lab but will likely suffer from resistance in the field.

This project focused on the design, synthesis, and testing of trifluoromethylphenyl amides (TFMPAs) as potential mosquitocides and repellents. Fourteen compounds were evaluated for toxicity against larvae and adults of Aedes aegypti. Several compounds were toxic against Aedes aegypti (larval, adult and feeding bioassays) and Drosophila melanogaster (glass-surface contact assay), but were much less toxic than fipronil, with toxicity ratios ranging from 100-fold in the larval assay to 100,000-fold for topical application to adult insects. In repellency bioassays to determine minimum effective dosage (MED), compound N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (7b) repelled Ae. aegypti females at lower concentration, 0.017 (±0.006) μmol/cm 2 , than N, N-diethyl-meta-toluamide (DEET) 0.026 (±0.005) μmol/cm 2 . 2-Chloro-N-(3-(trifluoromethyl)phenyl)acetamide (6a) performed better than DEET against two species of mosquitoes: it repelled Ae. aegypti females at 0.013 (±0.006) μmol/cm 2 and Anopheles gambiae females (in a warm body repellent assay), at a standard exposure of 2 nmol/cm 2 . These studies revealed novel active structures that could further lead to compounds with better repellent activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

G-Protein-Coupled Receptors (GPCRs) are an underdeveloped target in the search for agrochemicals with octopamine receptors, a GPCR, being the target of a single insecticide/acaricide class (formamidines). The evolution of insecticide resistance has resulted in the need to identify new or underutilized targets for the development of agrochemicals, with the goal of controlling arthropod pests that affect agriculture or human and animal health. The insect cholinergic system has been a fruitful target for the development of insecticides/acaricides viz. acetylcholinesterase inhibitors and agonists/modulators of the nicotinic acetylcholine receptor. However, the muscarinic acetylcholine receptors (mAChRs), which are GPCRs, have not been successfully developed as a target for agrochemicals. Others have recently identified three subtypes of insect mAChRs in Drosophila melanogaster, and extracellular recordings from transected D. melanogaster larval central nervous system (CNS) were performed to investigate the electrogenesis of the octopaminergic and muscarinic systems. Octopamine (10 μM) resulted in a sustained neuroexcitation during a 30 min exposure, and neuroexcitation after 21 min was blocked by octopamine receptor antagonist, phentolamine (100 μM). Exposure of this preparation to the non-selective mAChR agonist, pilocarpine (10 μM), resulted in a biphasic response, characterized by neuroexcitation followed by a decrease in the CNS firing rate below initial control levels. This biphasic effect was antagonized by the classical mAChR antagonist atropine (10 μM). It was also found that atropine (10 μM) blocked octopamine's sustained neuroexcitation, indicating the possibility of cross-talk between these two GPCR pathways., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study focused on the toxicity of the aphid anti-feedant flonicamid and its main metabolite, 4-trifluoromethylnicotinamide (TFNA-AM) to Aedes aegypti and Anopheles gambiae mosquitoes. The compounds were toxic to both species via topical application, resulting in un-coordinated locomotion and leg splaying, with a favorable An. gambiae LD 50 value of 35 ng/mg for TFNA-AM, but no significant lethality to Ae. aegypti at 10 μg/female. There was mild cross resistance in the Akron-kdr (Akdr) strain of An. gambiae. Both compounds were non-toxic to intact larvae (LC 50  > 300 ppm); however, headless Ae. aegypti larvae displayed spastic paralysis, with PC 50 values of 2-4 ppm, indicating that the cuticle is a significant barrier to penetration. TFNA-AM showed low mammalian toxicity, with an LD 50 of >2000 mg/kg in mice. Electrophysiological experiments showed larval Aedes muscle depolarization and Kv2 channel blocking activity that required near mM concentrations, suggesting that this potassium channel is not the main target for flonicamid nor its metabolite. However, TFNA-AM was a potent blocker of evoked body wall sensory discharge in dipteran larvae, suggesting that some component of the chordotonal organ system may be involved in its toxicity. Finally, flonicamid and TFNA-AM showed about 2-fold synergism of permethrin toxicity against An. gambiae adult females whose mechanism should become more clear once the mode of action of these compounds is better defined., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The widespread emergence of pyrethroid-resistant Anopheles gambiae has intensified the need to find new contact mosquitocides for indoor residual spraying and insecticide treated nets. With the goal of developing new species-selective and resistance-breaking acetylcholinesterase (AChE)-inhibiting mosquitocides, in this report we revisit the effects of carbamate substitution on aryl carbamates, and variation of the 1-alkyl group on pyrazol-4-yl methylcarbamates. Compared to aryl methylcarbamates, aryl dimethylcarbamates were found to have lower selectivity for An. gambiae AChE ( Ag AChE) over human AChE (hAChE), but improved tarsal contact toxicity to G3 strain An. gambiae . Molecular modeling studies suggest the lower species-selectivity of the aryl dimethylcarbamates can be attributed to a less flexible acyl pocket in Ag AChE relative to hAChE. The improved tarsal contact toxicity of the aryl dimethylcarbamates relative to the corresponding methylcarbamates is attributed to a range of complementary phenomena. With respect to the pyrazol-4-yl methylcarbamates, the previously observed low An. gambiae -selectivity of compounds bearing α-branched 1-alkyl groups was improved by employing β- and γ-branched 1-alkyl groups. Compounds 22a (cyclopentylmethyl), 21a (cyclobutylmethyl), and 26a (3-methylbutyl) offer 250-fold, 120-fold, and 96-fold selectivity, respectively, for inhibition of Ag AChE vs. hAChE. Molecular modeling studies suggests the high species-selectivity of these compounds can be attributed to the greater mobility of the W84 side chain in the choline-binding site of Ag AChE, compared to that of W86 in hAChE. Compound 26a has reasonable contact toxicity to G3 strain An. gambiae (LC 50 = 269 μg/mL) and low cross-resistance to Akron strain (LC 50 = 948 μg/mL), which bears the G119S resistance mutation., Competing Interests: Conflict of Interest: None of the authors have any conflict to report.

Horn flies, Haematobia irritans , a major cattle pest in the USA, cause substantial economic losses and current control methods rely heavily on insecticides. Three horn fly populations were evaluated for insecticide susceptibility to permethrin, &beta;-cyfluthrin, and diazinon. Susceptibility was variable by population, with the greatest resistance exhibited by a 66-fold resistance ratio (RR) to permethrin and >14-fold RR to diazinon. Mechanisms of resistance were determined using molecular techniques and enzymatic assays. The knockdown resistance ( kdr ) genotype (L150F) associated with pyrethroid resistance, and a G262A mutation in acetylcholinesterase, previously associated with organophosphate resistance, were found in all field populations evaluated. Insensitivity of diazoxon at the acetylcholinesterase (AChE) target site was significantly different in horn flies from one of the field sites. For metabolic detoxifying enzymes, cytochrome P450 nor general esterases showed a significant difference between field strains and a laboratory susceptible strain. Pyrethroid resistance was likely due to the presence of the L150F mutation in the population. In vitro studies targeting the AChE enzyme did not support the notion that the G262A mutation was the sole cause of resistance to organophosphates, and, therefore, the exact resistance mechanism to diazinon was not able to be confirmed.

Background: 1,3,4-Oxadiazole and imidazolidine rings are important heterocyclic compounds exhibiting a variety of biological activities. In this study, novel compounds with oxadiazole and imidazolidine rings were synthesized from 3-(methylsulfonyl)-2-oxoimidazolidine-1-carbonyl chloride and screened for insecticidal activities. The proposed structures of the 17 synthesized compounds were confirmed using elemental analysis, infrared (IR), proton nuclear magnetic resonance ( 1 H-NMR), and mass spectroscopy., Results: None of the compounds showed larvicidal activity at the tested concentrations against first-instar Aedes aegypti larvae. However, nine compounds exhibited promising adulticidal activity, with mortality rates of ≥80% at 5 µg per mosquito. Further dose-response bioassays were undertaken to determine median lethal dose (LD 50 ) values. Compounds 1, 2b, 2c, 2d, 2 g, 3b, 3c, 3 g, and 3 h were effective, with typical LD 50 values of about 5 - 10 µg per mosquito against female Ae. aegypti. Compounds 2c (bearing a nitro group on the aromatic ring; LD 50 = 2.80 ± 0.54 µg per mosquito) and 3 h (double halogen groups at 2,4 position on the phenyl ring; LD 50 = 2.80 ± 0.54 µg per mosquito) were the most promising compounds., Conclusion: Preliminary mode of action studies failed to show consistent evidence of either neurotoxic or mitochondria-directed effects. Further chemical synthesis within this series may lead to the development of new effective insecticides. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)