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Inward rectifier K+ (Kir) channels have been studied extensively in mammals, where they play critical roles in health and disease. In insects, Kir channels have recently been found to be key regulators of diverse physiological processes in several tissues. The importance of Kir channels in insects has positioned them to serve as emerging targets for the development of insecticides with novel modes of action. In this article, we provide the first comprehensive review of insect Kir channels, highlighting the rapid progress made in understanding their molecular biology, physiological roles, pharmacology, and toxicology. In addition, we highlight key gaps in our knowledge and suggest directions for future research to advance our understanding of Kir channels and their roles in insect physiology. Further knowledge of their functional roles will also facilitate their exploitation as targets for controlling arthropod pests and vectors of economic, medical, and/or veterinary relevance. Expected final online publication date for the Annual Review of Entomology, Volume 67 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

We previously extracted and purified a chromene amide from Amyris texana and found this scaffold is moderately insecticidal and thus, this study aimed to test the insecticidal properties of 13 synthetically derived chromene analogs to the fall armyworm (FAW, Spodoptera frugiperda). Microinjection of chromenes with alcohol or aldehydes substitutions at the meta position on the benzopyran moiety led to moderate toxicity that was approximately 2- to 3-fold less toxic when compared to permethrin, yet microinjection of differently substituted chromenes exhibited little to no toxicity. Similarly, chromenes with alcohol or aldehydes substitutions at the meta position on the benzopyran moiety were among the most toxic chromenes studied through ingested exposure. In addition to acute toxicity, select chromenes significantly increased the percentage of developmental defects upon eclosion that prevented adult moths from being capable of flight, suggesting these compounds alter development. Interestingly, microinjection yielded differing signs of intoxication between alcohol and aldehyde substitutions where the alcohol resulted in flaccid paralysis and lethargy whereas aldehyde led to tonic contractions and hyperactivity. These contrasting signs of intoxication were also observed in electrophysiological assays where alcohol substitutions led to the depression of central neuron firing activity and aldehyde substitutions led to hyperexcitation of central neurons. In summary, the chromene amides led to acute lethality and/or altered developmental trajectories of FAW, yet the high doses required for acute mortality suggest these scaffolds hold relatively little promise for development into FAW-directed insecticides but may represent novel growth regulators for FAW.

The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a destructive agricultural pest, capable of photosynthate removal and plant virus transmission. Therefore, we aimed to test the antifeedant properties of small-molecule inhibitors of inward rectifier potassium (Kir) channels expressed in insect salivary glands and develop an approach for enabling systemic movement of lipophilic Kir inhibitors.Two Kir channel inhibitors, VU041 and VU730, reduced the secretory activity of the aphid salivary glands by 3.3-fold and foliar applications of VU041 and VU730 significantly (P 0.05) increased the time to first probe, total probe duration, and nearly eliminated phloem salivation and ingestion. Next, we aimed to facilitate systemic movement of VU041 and VU730 through evaluation of a novel natural product based solubilizer containing rubusoside that was isolated from Chinese sweet leaf (Rubus suavissimus) plants. A single lower leaf was treated with Kir inhibitor soluble liquid (KI-SL) and systemic movement throughout the plant was verified via toxicity bioassays and changes to feeding behavior through the electrical penetration graph (EPG) technique. EPG data indicate KI-SL significantly reduced ability to reach E1 (phloem salivation) and E2 (phloem ingestion) waveforms and altered plant probing behavior when compared to the untreated control. High-performance liquid chromatography (HPLC) analysis indicated the presence of VU041 and VU730 in the upper leaf tissue of these plants. Together, these data provide strong support that incorporation of rubusoside with Kir inhibitors enhanced translaminar and translocation movement through the plant tissue.These data further support hemipteran Kir channels as a target to prevent feeding and induce toxicity. Further, these studies highlight a novel delivery approach for generating plant systemic activity of lipophilic insecticides. © 2022 Society of Chemical Industry.

Background The loss of honey bee colonies is a nationally recognized problem that demands attention from both the scientific community and the beekeeping industry. One outstanding threat is the unintended exposure of these pollinators to agricultural pesticides. Anthranilic diamides, such as chlorantraniliprole, are registered for use in stone and pome fruits, vegetables, turf, and grains. There are few publicly available studies that provide an analysis of chlorantraniliprole effects on the survivorship and locomotion activity of beneficial, pollinating insects such as honey bees. The data gathered in this study provide the acute toxicity, 30-day survivorship, and locomotor activity of honey bees exposed to technical-grade chlorantraniliprole and three formulated products with chlorantraniliprole as the active ingredient. Results Neither the technical-grade nor the formulated products of chlorantraniliprole were acutely toxic to honey bees following 4 or 72h treatments at the tested concentrations. A 4 h treatment of technical-grade and formulated chlorantraniliprole did not significantly affect the 30-day survivorship, although significantly higher mortality was observed after 30 days for bees receiving a 72 h treatment of technical-grade chlorantraniliprole and two formulated products. The locomotion activity, or total walking distance, of bees receiving a 4 h treatment of one chlorantraniliprole formulation was significantly reduced, with these individuals recovering their normal locomotion activity at 48 h post exposure. Conversely, there was observed lethargic behavior and significantly reduced walking distances for bees provided with a 72 h treatment of technical-grade chlorantraniliprole and each formulated product. Conclusion This study provides evidence for the effect of long-term exposure of chlorantraniliprole on the survivorship and locomotor activity of honey bees. Bees receiving a more field-relevant short-term exposure survived and moved similarly to untreated bees, reiterating the relative safety of chlorantraniliprole exposure to adult honey bees at recommended label concentrations. © 2020 Society of Chemical Industry.

The cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is one of the most destructive agricultural pests due to photosynthate removal and horizontal transmission of plant viruses. Horizontal transmission of plant viruses by aphids occurs during distinct feeding behavioral events, such as probing for non-persistent viruses or phloem feeding for persistent viruses. We employed toxicity bioassays and electrical penetration graph (EPG) methodology to compare toxicity and quantify changes to feeding behavior and toxicity of A. gossypii after exposure to commercialized aphicides. Commercialized aphicides containing flupyradifurone, sulfoxaflor, thiamethoxam, thiamethoxam + lambda cyhalothrin, and bifenthrin induced90% aphid mortality within 4 h of exposure. Flupyradifurone was the most acutely toxic aphicide studied with an LT

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

The acute toxicity of chlorpyrifos and chlorpyrifos-oxon (organophosphorothioate insecticides) was examined alone and in combination with atrazine (triazine herbicide) and alachlor (chloroacetanilide herbicide) to honey bees (Apis mellifera). Atrazine and alachlor were observed to not be acutely toxic to bees at doses up to 10 and 4 μg per bee, respectively. However, atrazine significantly increased chlorpyrifos toxicity by 3-fold while reducing chlorpyrifos-oxon toxicity by 1.8-fold. These changes in toxicity are correlated with significant 1.3- and 1.2-fold inhibition of acetylcholinesterase (AChE) activity in bees exposed to chlorpyrifos and chlorpyrifos-oxon, respectively. Atrazine significantly increased cytochrome P450, general esterase, and glutathione S-transferase (GST) activities by 1.5-, 1.2-, and 1.2- fold respectively, in bees compared to untreated individuals. Alachlor increased chlorpyrifos toxicity by 2.5-fold but did not affect the toxicity of chlorpyrifos-oxon. Exposure to alachlor and chlorpyrifos did not affect AChE compared to chlorpyrifos alone. However, exposure to chlorpyrifos-oxon and alachlor significantly increased acetylcholinesterase (AChE) activity by 1.4-fold. GST activity, but not P450 or general esterases, was significantly increased in bees exposed to alachlor. These data provide evidence that triazine and chloroacetanilide herbicide exposure alters detoxification enzyme activity and, in turn, alters the sensitivity of bees to organophosphorothioate insecticides. Importantly, these data can be used to guide future studies aiming to test safety profiles for pollinators and expand regulatory framework required for pesticide registration.

Resistance mechanisms to synthetic insecticides often include point mutations and increased expression of genes encoding detoxification enzymes. Since pyrethroids are the main adulticides used against Aedes aegypti, which vectors pathogens such as Zika virus, understanding resistance to this insecticide class is of significant relevance. We focused on adenosine triphosphate (ATP)-binding cassette (ABC) transporters in the pyrethroid-resistant Puerto Rico (PR) strain of Ae. aegypti. We investigated the expression patterns of six ABC transporters previously characterized as differentially expressed in insecticide-challenged mosquitoes, or increased mRNA expression in pyrethroid-resistant Ae. aegypti, by comparing PR to the Rockefeller (Rock) susceptible strain. No constitutive differential expression between strains was detected, but expression differences for these genes was influenced by sex and age, suggesting that their role is independent from resistance in PR. Instead, ABC transporters may be induced after insecticide exposure. Challenging mosquitoes with deltamethrin, with or without ABC transporter modulators, showed that Rock and PR responded differently, but a contribution of ABC transporters to deltamethrin toxicity is suspected. Moreover, the effect of dexamethasone, which enhanced the inhibition of nerve firing by deltamethrin, was observed using a Drosophila central nervous system preparation, showing synergy of these two compounds through the potential inhibition of ABC transporters.

GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K+-Cl- cotransporter (KCC) is the primary mechanism by which mature neurons maintain low intracellular Cl- concentration, yet the fundamental physiology, comparative physiology, and toxicological relevance of insect KCC is understudied. Considering this, we employed electrophysiological, genetic, and pharmacological methods to characterize the physiological underpinnings of KCC function to the Drosophila CNS. Our data show that genetic ablation or pharmacological inhibition of KCC results in an increased spike discharge frequency and significantly ( P < 0.05) reduces the CNS sensitivity to γ-aminobutyric acid (GABA). Further, simultaneous inhibition of KCC and ligand-gated chloride channel (LGCC) complex results in a significant ( P < 0.001) increase in CNS spontaneous activity over baseline firing rates that supports functional coupling of KCC to LGCC function. Interestingly, 75% reduction in KCC mRNA did not alter basal neurotransmission levels indicating that only a fraction of the KCC population is required to maintain the Cl- ionic gradient when at rest, but prolonged synaptic activity increases the threshold for GABA-mediated inhibition and reduces nerve sensitivity to GABA. These data expand current knowledge regarding the physiological role of KCC in a model insect and provides the necessary foundation to develop KCC as a novel biochemical target of insecticides, as well as complements existing research to provide a holistic understanding of the plasticity in mammalian health and disease.

Background The unique feeding biology of aphids suggests novel insecticide targets are likely to exist outside of the nervous system. We therefore aimed to directly test the hypothesis that pharmacological inhibition of inward rectifier potassium (Kir) channels would result in salivary gland failure and reduced sap ingestion by the cotton aphid, Aphis gossypii. Results The Kir inhibitors VU041 and VU590 reduced the length of the salivary sheath in a concentration dependent manner, indicating that the secretory activity of the salivary gland is reduced by Kir inhibition. Next, we employed the electrical penetration graph (EPG) technique to measure the impact Kir inhibition has to aphid sap feeding and feeding biology. Data show that foliar application of VU041 eliminated the E1 and E2 phases (phloem feeding) in all aphids studied. Contact exposure to VU041 after foliar applications was found to be toxic to A. gossypii at 72 and 96 h post-infestation, indicating mortality is likely a result of starvation and not acute toxicity. Furthermore, VU041 exposure significantly altered the feeding behavior of aphids, which is toxicologically relevant for plant-virus interactions. Conclusion These data suggest Kir channels are critical for proper function of aphid salivary glands and the reduced plant feeding justifies future work in developing salivary gland Kir channels as novel mechanism aphicides. Furthermore, products like VU041 would add to a very minor arsenal of compounds that simultaneously reduce vector abundance and alter feeding behavior. © 2019 Society of Chemical Industry.

The majority of commercialized insecticides target the insect nervous system and therefore, neural proteins are well-validated targets for insecticide development. Considering that only a few neural targets are exploited for insecticidal action and the development of insecticide resistance has reduced the efficacy of current insecticidal classes, we sought to test the toxicological potential of the potassium-chloride cotransporter (KCC). In mammals, KCC proteins have seminal roles in shaping GABAergic signaling and inhibitory neurotransmission, thus ion transport through KCC is critical for proper neurotransmission. Therefore, we hypothesized that mosquito KCC represents a putative insecticide target site and that pharmacological inhibition of KCC constructs in Aedes aegypti will be lethal. To test this hypothesis, we developed a robust, cell-based fluorescence assay that enables in vitro characterization of small-molecules against Ae. aegypti KCC and performed a proof-of-concept study employing well characterized mammalian KCC modulators to determine the toxicological potential of Ae. aegypti KCC. The selective inhibitor of mammalian KCC, termed VU0463271, was found to be a potent inhibitor Ae. aegypti KCC and microinjection induced lethality in a concentration-dependent manner to susceptible and pyrethroid resistant strains. Importantly, an analog of VU0463271 was shown to be >40-fold less potent and did not induce toxicity, suggesting that the observed physiological effects and mortality are likely due to KCC inhibition. This proof-of-concept study suggests that Ae. aegypti KCC represents a putative target site for mosquitocide design that can mitigate the current mechanisms of insecticide resistance.

The fall armyworm (FAW), Spodoptera frugiperda, is a global pest of multiple economically important row crops and the development of resistance to commercially available insecticidal classes has inhibited FAW control. Thus, there is a need to identify chemical scaffolds that can provide inspiration for the development of novel insecticides for FAW management. This study aimed to assess the sensitivity of central neurons and susceptibility of FAW to chloride channel modulators to establish a platform for repurposing existing insecticides or designing new chemicals capable of controlling FAW. Potency of select chloride channel modulators were initially studied against FAW central neuron firing rate and rank order of potency was determined to be fipronil > lindane > Z-stilbene > DIDS > GABA > E-stilbene. Toxicity bioassays identified fipronil and lindane as the two most toxic modulators studied with topical LD50's of 41 and 75 ng/mg of caterpillar, respectively. Interestingly, Z-stilbene was toxic at 300 ng/mg of caterpillar, but no toxicity was observed with DIDS or E-stilbene. The significant shift in potency between stilbene isomers indicates structure-activity relationships between stilbene chemistry and the binding site in FAW may exist. The data presented in this study defines the potency of select chloride channel modulators to FAW neural activity and survivorship to establish a platform for development of novel chemical agents to control FAW populations. Although stilbenes may hold promise for insecticide development, the low toxicity of the scaffolds tested in this study dampen enthusiasm for their development into FAW specific insecticides.

The arthropod salivary gland is of critical importance for horizontal transmission of pathogens, yet a detailed understanding of the ion conductance pathways responsible for saliva production and excretion is lacking. A superfamily of potassium ion channels, known as inward rectifying potassium (Kir) channels, is overexpressed in the Drosophila salivary gland by 32-fold when compared to the whole body mRNA transcripts. Therefore, we aimed to test the hypothesis that pharmacological and genetic depletion of salivary gland specific Kir channels alters the efficiency of the gland and reduced feeding capabilities using the fruit fly Drosophila melanogaster as a model organism that could predict similar effects in arthropod disease vectors. Exposure to VU041, a selective Kir channel blocker, reduced the volume of sucrose consumption by up to 3.2-fold and was found to be concentration-dependent with an EC50 of 68μM. Importantly, the inactive analog, VU937, was shown to not influence feeding, suggesting the reduction in feeding observed with VU041 is due to Kir channel inhibition. Next, we performed a salivary gland specific knockdown of Kir1 to assess the role of these channels specifically in the salivary gland. The genetically depleted fruit flies had a reduction in total volume ingested and an increase in the time spent feeding, both suggestive of a reduction in salivary gland function. Furthermore, a compensatory mechanism appears to be present at day 1 of RNAi-treated fruit flies, and is likely to be the Na+-K+-2Cl− cotransporter and/or Na+-K+-ATPase pumps that serve to supplement the inward flow of K+ ions, which highlights the functional redundancy in control of ion flux in the salivary glands. These findings suggest that Kir channels likely provide, at least in part, a principal potassium conductance pathway in the Drosophila salivary gland that is required for sucrose feeding.

Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. In the work described here, we have demonstrated the use of flock house virus (FHV) as a model system for virus infection in bees and revealed an important role for the regulation of the bee antiviral immune response by ATP-sensitive inwardly rectifying potassium (KATP) channels. We have shown that treatment with the KATP channel agonist pinacidil increases survival of bees while decreasing viral replication following infection with FHV, whereas treatment with the KATP channel antagonist tolbutamide decreases survival and increases viral replication. Our results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees.

The salivary gland of hematophagous arthropods is critical for blood meal acquisition, blood vessel localization, and secretion of digestive enzymes. Thus, there is significant interest in the regulation of salivary gland function and mechanisms driving the secretion of saliva and digestive proteins. We aimed to gain a broader understanding of the regulatory role of aminergic, cholinergic, and octopaminergic neuromodulators to saliva and protein secretion from the female A. aegypti salivary gland. Quantification of saliva after injection with neuromodulators showed that dopamine, serotonin, and pilocarpine increased the secretory activity of the salivary gland with potency rankings dopamine = serotonin > pilocarpine. No change in saliva secretion was observed with octopamine or ergonovine, which indicates the A. aegypti salivary gland may be regulated by dopaminergic, serotonergic, and cholinergic systems, but are not likely regulated by octopaminergic or tryptaminergic systems. Next, we studied the regulatory control of dopamine-mediated salivation. Data indicate extracellular calcium flux, but not neural function, is critical for dopamine-mediated salivation, which suggests epithelial transport of ions and not neuronal control is responsible for dopamine-mediated salivation. For regulation of protein secretion, data indicate dopamine or serotonin exposure facilitates amylase secretion, whereas serotonin but not dopamine exposure increased apyrase concentrations in the secreted saliva. General immunoreactivity to anti-rat D1-dopamine receptor antibody was observed, yet immunoreactivity to the anti-rat D2-receptor antibody was identified in the proximal regions of the lateral lobes and slight immunoreactivity in the distal portion of the lateral lobe, with no expression in the medial lobe.

Neurophysiological recordings were employed to quantify neuronal sensitivity to neurotoxic insecticides and assessed toxicity across field and laboratory fall armyworm (FAW) populations. Topical toxicity resistance ratios (RR) in field-collected FAW was 767-fold compared to laboratory strains and, importantly, a 1750-fold reduction in potency was observed for λ-cyhalothrin in neurophysiological assays. Field collected FAW were found to have a RR of 12 to chlorpyrifos when compared to the susceptible strain and was 8-fold less sensitive in neurophysiological assays. Surprisingly, there were no point mutations identified in the voltage-gated sodium channel known to cause pyrethroid resistance. For acetylcholinesterase, FAW had more than 80% of their nucleotide sequences consistent with A201 and F290 of the susceptible strains although 60% of the tested population was heterozygous for the G227A mutation. These data indicate that point mutations did not contribute to the high level of pyrethroid resistance and nerve insensitivity in this population of field collected FAW. Additionally, these data suggest the kdr phenotype only explains a portion of the heritable variation in FAW resistance and indicates kdr is not the only predictor of high pyrethroid resistance. Phenotypic assays, such as toxicity bioassays or neurophysiological recordings, using field-collected populations are necessary to reliably predict resistant phenotypes and product failures.

Bloodmeal feeding by the horn fly, Haematobia irritans (L.), is associated with reduced milk production and blood loss that ultimately prevents weight gain of calves and yearlings. Thus, blood feeding by H. irritans causes significant economic losses in several continents. As with other arthropods, resistance to the majority of commercialized insecticides reduces the efficacy of current control programs. Thus, innovative technologies and novel biochemical targets for horn fly control are needed. Salivary gland and Malpighian tubule function are critical for H. irritans survivorship as they drive bloodmeal acquisition and maintain ion- and fluid homeostasis during bloodmeal processing, respectively. Experiments were conducted to test the hypothesis that pharmacological modulation of H. irritans inward rectifier potassium (Kir) channels would preclude blood feeding and induce mortality by reducing the secretory activity of the salivary gland while simultaneously inducing Malpighian tubule failure. Experimental results clearly indicate structurally diverse Kir channel modulators reduce the secretory activity of the salivary gland by up to fivefold when compared to control and the reduced saliva secretion was highly correlated to a reduction in bloodmeal acquisition in adult flies. Furthermore, adult feeding on blood treated with Kir channel modulators resulted in significant mortality. In addition to validating the Kir channels of H. irritans as putative insecticide targets, the knowledge gained from this study could be applied to develop novel therapeutic technologies targeting salivary gland or Malpighian tubule function to reduce the economic burden of horn fly ectoparasitism on cattle health and production.

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.

The discovery and development of new active ingredients to control arthropod populations and circumvent the inevitable evolution of insecticide resistance has been of consistent interest to the field of insecticide science. This interest has resulted in a slow, but steady increase in the diversity of chemical scaffolds and biochemical target sites within the insecticide arsenal over the past 70 years with growth from three biochemical target sites in the 1950s to 22 distinct biochemical targets in 2018. Despite this growth, the number of biochemical target sites for insecticides remains relatively limited when compared to human pharmaceuticals, which has approximately 700 distinct biochemical targets that are targeted by FDA approved drugs. Potential reasons for this large discrepancy between two closely related fields and putative mechanisms to enhance the identification of tractable biochemical targets for insecticides are discussed. Next, this perspective discusses the movement of insecticide science into the “genomic era” and for comparative purposes, I provide a retrospective analysis of the impact the release of the human genome had to human pharmaceutical development. Based on this analysis and because the fields of insecticide science and human pharmaceuticals mirror each other, researchers in the field of insecticide science would do well to heed the lessons learned by the human pharmaceutical industry and to carefully consider the challenges that arise from genomic approaches for chemical development. Lastly, I pose the question if the field of insecticide science would benefit from adapting an industry-academia model through the generation of industry-sponsored centers of excellence. The goal of this article is not to definitively describe strategies to enhance insecticide development, but rather present different thoughts on agrochemical development that will foster discussions among academic, government, and industry scientists to address current and future problems in the field of insecticide science.

GABAergic signaling is the cornerstone for fast synaptic inhibition of neural signaling in arthropods and mammals and is the molecular target for insecticides and pharmaceuticals, respectively. The K

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.

Mosquitoes use chemical cues to modulate important behaviors such as feeding, mating, and egg laying. The primary chemosensory organs comprising the paired antennae, maxillary palps and labial palps are adorned with porous sensilla that house primary sensory neurons. Dendrites of these neurons provide an interface between the chemical environment and higher order neuronal processing. Diverse proteins located on outer membranes interact with chemicals, ions, and soluble proteins outside the cell and within the lumen of sensilla. Here, we review the repertoire of chemosensory receptors and other membrane proteins involved in transduction and discuss the outlook for their functional characterization. We also provide a brief overview of select ion channels, their role in mammalian taste, and potential involvement in mosquito taste. These chemosensory proteins represent targets for the disruption of harmful biting behavior and disease transmission by mosquito vectors.

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 &gt, 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.

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein–coupled inwardly rectifying potassium (GIRK) channels by G protein–coupled receptors (GPCRs) via the G protein βγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1.

Insecticide resistance in the malaria vector, Anopheles gambiae, is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a "pseudo-pyrethroid." There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety.

A complete understanding of the physiological pathways critical for proper function of the insect nervous system is still lacking. The recent development of potent and selective small-molecule modulators of insect inward rectifier potassium (Kir) channels has enabled the interrogation of the physiological role and toxicological potential of Kir channels within various insect tissue systems. Therefore, we aimed to highlight the physiological and functional role of neural Kir channels the central nervous system, muscular system, and neuromuscular system through pharmacological and genetic manipulations. Our data provide significant evidence that Drosophila neural systems rely on the inward conductance of K+ ions for proper function since pharmacological inhibition and genetic ablation of neural Kir channels yielded dramatic alterations of the CNS spike discharge frequency and broadening and reduced amplitude of the evoked EPSP at the neuromuscular junction. Based on these data, we conclude that neural Kir channels in insects (1) are critical for proper function of the insect nervous system, (2) represents an unexplored physiological pathway that is likely to shape the understanding of neuronal signaling, maintenance of membrane potentials, and maintenance of the ionic balance of insects, and (3) are capable of inducing acute toxicity to insects through neurological poisoning.

The majority of commercialized insecticides target the insect nervous system and therefore, neural proteins are well-validated targets for insecticide development. Considering that only a few neural targets are exploited for insecticidal action and the development of insecticide resistance has reduced the efficacy of current insecticidal classes, we sought to test the toxicological potential of the potassium-chloride cotransporter (KCC). In mammals, KCC proteins have seminal roles in shaping GABAergic signaling and inhibitory neurotransmission, thus ion transport through KCC is critical for proper neurotransmission. Therefore, we hypothesized that mosquito KCC represents a putative insecticide target site and that pharmacological inhibition of KCC constructs in Aedes aegypti will be lethal. To test this hypothesis, we developed a robust, cell-based fluorescence assay that enables in vitro characterization of small-molecules against Ae. aegypti KCC and performed a proof-of-concept study employing well characterized mammalian KCC modulators to determine the toxicological potential of Ae. aegypti KCC. The selective inhibitor of mammalian KCC, termed VU0463271, was found to be a potent inhibitor Ae. aegypti KCC and microinjection induced lethality in a concentration-dependent manner to susceptible and pyrethroid resistant strains. Importantly, an analog of VU0463271 was shown to be40-fold less potent and did not induce toxicity, suggesting that the observed physiological effects and mortality are likely due to KCC inhibition. This proof-of-concept study suggests that Ae. aegypti KCC represents a putative target site for mosquitocide design that can mitigate the current mechanisms of insecticide resistance.

BACKGROUND Insecticide resistance in the malaria mosquito Anopheles gambiae is well documented, and widespread agricultural use of pyrethroids may exacerbate development of resistance when pyrethroids are used in vector control. We have developed carbamate anticholinesterases that possess a high degree of An. gambiae:human selectivity for enzyme inhibition. The purpose of this study was to assess the spectrum of activity of these carbamates against other mosquitoes and agricultural pests. RESULTS Experimental carbamates were potent inhibitors of mosquito acetylcholinesterases, with IC50 values in the nanomolar range. Similar potencies were observed for Musca domestica and Drosophila melanogaster enzymes. Although meta-substituted carbamates were potent inhibitors, two ortho-substituted carbamates displayed poor enzyme inhibition (IC50 ≥ 10−6 M) in honey bee (Apis mellifera), Asian citrus psyllid (Diaphorina citri) and lepidopteran agricultural pests (Plutella xylostella and Ostrinia nubilalis). Enzyme inhibition results were confirmed by toxicity studies in caterpillars, where the new carbamates were 2–3-fold less toxic than propoxur and up to tenfold less active than bendiocarb, indicating little utility of these compounds for crop protection. CONCLUSION The experimental carbamates were broadly active against mosquito species but not agricultural pests, which should mitigate selection for mosquito insecticide resistance by reducing agricultural uses of these compounds. © 2014 Society of Chemical Industry

Inward rectifier potassium (Kir) channels play fundamental roles in cardiac and renal function and may represent unexploited drug targets for cardiovascular diseases. However, the limited pharmacology of Kir channels has slowed progress toward exploring their integrative physiology and therapeutic potential. Here, we review recent progress toward developing the small-molecule pharmacology for Kir2.x, Kir4.1, and Kir7.1 and discuss common mechanistic themes that may help guide future Kir channel-directed drug discovery efforts.

Twenty trifluoromethylphenyl amides were synthesized and evaluated as fungicides and as mosquito toxicants and repellents. Against Aedes aegypti larvae, N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-3,5-dinitrobenzamide (1e) was the most toxic compound (24 h LC50 1940 nM), while against adults N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,2-trifluoroacetamide (1c) was most active (24 h LD50 19.182 nM, 0.5 μL/insect). However, the 24 h LC50 and LD50 values of fipronil against Ae. aegypti larvae and adults were significantly lower: 13.55 nM and 0.787 × 10(-4) nM, respectively. Compound 1c was also active against Drosophila melanogaster adults with 24 h LC50 values of 5.6 and 4.9 μg/cm(2) for the Oregon-R and 1675 strains, respectively. Fipronil had LC50 values of 0.004 and 0.017 μg/cm(2) against the two strains of D. melanogaster, respectively. In repellency bioassays against female Ae. aegypti, 2,2,2-trifluoro-N-(2-(trifluoromethyl)phenyl)acetamide (4c) had the highest repellent potency with a minimum effective dosage (MED) of 0.039 μmol/cm(2) compared to DEET (MED of 0.091 μmol/cm(2)). Compound N-(2-(trifluoromethyl)phenyl)hexanamide (4a) had an MED of 0.091 μmol/cm(2) which was comparable to DEET. Compound 4c was the most potent fungicide against Phomopsis obscurans. Several trends were discerned between the structural configuration of these molecules and the effect of structural changes on toxicity and repellency. Para- or meta- trifluoromethylphenyl amides with an aromatic ring attached to the carbonyl carbon showed higher toxicity against Ae. aegypti larvae, than ortho- trifluoromethylphenyl amides. Ortho- trifluoromethylphenyl amides with trifluoromethyl or alkyl group attached to the carbonyl carbon produced higher repellent activity against female Ae. aegypti and Anopheles albimanus than meta- or para- trifluoromethylphenyl amides. The presence of 2,6-dichloro- substitution on the phenyl ring of the amide had an influence on larvicidal and repellent activity of para- trifluoromethylphenyl amides.

International audience; We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist alpha-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of alpha-bungarotoxin-sensitive receptors and did not involve alpha-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.

Malaria is an urgent world health concern and vector control is one important option for reducing disease prevalence. Increased reports of pyrethroid-resistant mosquito strains have amplified the need for new vector-control chemicals. We compared three commercially available carbamate insecticides (carbofuran, bendiocarb, and propoxur) to eight experimental compounds 1–8 for activity against Anopheles gambiae acetylcholinesterase, as well as enzymes from mammalian, avian, and aquatic species. The experimental compounds (except 7) were less potent than the commercial inhibitors against the mosquito enzyme, but had higher selectivity values (up to near 600-fold, IC50 of non-target species/IC50 An. gambiae) because of their low potency for acetylcholinesterases from nontarget species. Neurotoxic esterase assay showed that none of the experimental carbamates (1 mM) displayed NTE inhibition, while bendiocarb did (24% inhibition at 1 mM), although the effect was much less than that of mipafox. In vivo bioassays using Daphnia magna showed that all novel carbamates were of similar killing potency as bendiocarb (24 h LC50 = 611 nM), with the exception of experimental compound 1 (LC50 = 172 nM). Overall, the results suggested that the novel carbamate insecticides 4–8 presented in this study were safer to mammals than the commercial compounds and were promising insecticides for malaria vector control usage on bednets or indoor residual sprays.

The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 μM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control.

ATP-sensitive inwardly rectifying potassium (KATP) channels couple cellular metabolism to the membrane potential of the cell and play an important role in a variety of tissue types, including the insect dorsal vessel, making them a subject of interest not only for understanding invertebrate physiology, but also as a potential target for novel insecticides. Most of what is known about these ion channels is the result of work performed in mammalian systems, with insect studies being limited to only a few species and physiological systems. The goal of this study was to investigate the role that KATP channels play in regulating cardiac function in a model social insect, the honey bee (Apis mellifera), by examining the effects that modulators of these ion channels have on heart rate. Heart rate decreased in a concentration-dependent manner, relative to controls, with the application of the KATP channel antagonist tolbutamide and KATP channel blockers barium and magnesium, whereas heart rate increased with the application of a low concentration of the KATP channel agonist pinacidil, but decreased at higher concentrations. Furthermore, pretreatment with barium magnified the effects of tolbutamide treatment and eliminated the effects of pinacidil treatment at select concentrations. The data presented here confirm a role for KATP channels in the regulation of honey bee dorsal vessel contractions and provide insight into the underlying physiology that governs the regulation of bee cardiac function.

Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects.

The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and non-selective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified Glutamate 149 and Alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 µM and Tmax = 3 hours) and favorable CNS distribution (mouse brain:plasma Kp of 10.9 to support in vivo studies for in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.

The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10μM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility.