Thermal infrared directs host-seeking behaviour in Aedes aegypti mosquitoes.

Mosquito-borne diseases affect hundreds of millions of people annually and disproportionately impact the developing world1,2. One mosquito species, Aedes aegypti, is a primary vector of viruses that cause dengue, yellow fever and Zika. The attraction of Ae. aegypti female mosquitos to humans requires integrating multiple cues, including CO2 from breath, organic odours from skin and visual cues, all sensed at mid and long ranges, and other cues sensed at very close range3-6. Here we identify a cue that Ae. aegypti use as part of their sensory arsenal to find humans. We demonstrate that Ae. aegypti sense the infrared (IR) radiation emanating from their targets and use this information in combination with other cues for highly effective mid-range navigation. Detection of thermal IR requires the heat-activated channel TRPA1, which is expressed in neurons at the tip of the antenna. Two opsins are co-expressed with TRPA1 in these neurons and promote the detection of lower IR intensities. We propose that radiant energy causes local heating at the end of the antenna, thereby activating temperature-sensitive receptors in thermosensory neurons. The realization that thermal IR radiation is an outstanding mid-range directional cue expands our understanding as to how mosquitoes are exquisitely effective in locating hosts.

Pymetrozine activates TRPV channels of brown planthopper Nilaparvata lugens.

The commercial insecticide pymetrozine has been extensively used for brown planthopper control in East Asia. The transient receptor potential vanilloid (TRPV) channel, which consists of two proteins, Nanchung (Nan) and Inactive (Iav), has recently been shown to be the molecular target of pymetrozine in the fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum). In this study, we characterized the Nan and Iav TRPV channel subunits of N. lugens and measured the action of pymetrozine on them. NlNan and NlIav are structurally similar to homologs from other insects. The expression pattern analysis of various body parts showed that NlNan and NlIav were both more abundantly expressed in antennae. When NlNan and NlIav were co-expressed in Xenopus laevis oocytes, they formed channels with high sensitivity to pymetrozine (EC50 = 5.5 × 10-8 M). Behavioral observation revealed that the gravitaxis defect in the fruit fly nan36a mutant was rescued by ectopically expressed NlNan and the rescued behavior could be abolished by pymetrozine. Our results confirm that NlNan and NlIav co-expressed complexes can be activated by pymetrozine both in vitro and in vivo and provide useful information for future resistance mechanism studies.

The novel pyridazine pyrazolecarboxamide insecticide dimpropyridaz inhibits chordotonal organ function upstream of TRPV channels.

BACKGROUND: Pyridazine pyrazolecarboxamides (PPCs) are a novel insecticide class discovered and optimized at BASF. Dimpropyridaz is the first PPC to be submitted for registration and controls many aphid species as well as whiteflies and other piercing-sucking insects. RESULTS: Dimpropyridaz and other tertiary amide PPCs are proinsecticides that are converted in vivo into secondary amide active forms by N-dealkylation. Active secondary amide metabolites of PPCs potently inhibit the function of insect chordotonal neurons. Unlike Group 9 and 29 insecticides, which hyperactivate chordotonal neurons and increase Ca2+ levels, active metabolites of PPCs silence chordotonal neurons and decrease intracellular Ca2+ levels. Whereas the effects of Group 9 and 29 insecticides require TRPV (Transient Receptor Potential Vanilloid) channels, PPCs act in a TRPV-independent fashion, without compromising cellular responses to Group 9 and 29 insecticides, placing the molecular PPC target upstream of TRPVs. CONCLUSIONS: PPCs are a new class of chordotonal organ modulator insecticide for control of piercing-sucking pests. Dimpropyridaz is a PPC proinsecticide that is activated in target insects to secondary amide forms that inhibit the firing of chordotonal organs. The inhibition occurs at a site upstream of TRPVs and is TRPV-independent, providing a novel mode of action for resistance management. © 2023 BASF Corporation. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Flonicamid metabolite 4-trifluoromethylnicotinamide is a chordotonal organ modulator insecticide†.

BACKGROUND: The selective aphicide flonicamid is known to cause symptoms in aphids that are like those of chordotonal organ TRPV channel modulator insecticides such as pymetrozine, pyrifluquinazon and afidopyropen. Flonicamid is classified by the Insecticide Resistance Action Committee as a chordotonal organ modulator with an undefined target site. However, although it has been shown not to act on TRPV channels, flonicamid's action on chordotonal organs has not been documented in the literature. RESULTS: Flonicamid causes locusts to extend their hindlegs, indicating an action on the femoral chordotonal organ. In fruit flies, it abolishes negative gravitaxis behavior by disrupting transduction and mechanical amplification in antennal chordotonal neurons. Although flonicamid itself only weakly affects locust chordotonal organs, its major animal metabolite 4-trifluoromethylnicotinamide (TFNA-AM) potently stimulates both locust and fly chordotonal organs. Like pymetrozine, TFNA-AM rapidly increases Ca2+ in antennal chordotonal neurons in wild-type flies, but not iav1 mutants, yet the effect is nonadditive with the TRPV channel agonist. CONCLUSIONS: Flonicamid is a pro-insecticide form of TFNA-AM, a potent chordotonal organ modulator. The functional effects of TFNA-AM on chordotonal organs of locusts and flies are indistinguishable from those of the TRPV agonists pymetrozine, pyrifluquinazon and afidopyropen. Because our previous results indicate that TFNA-AM does not act directly on TRPV channels, we conclude that it acts upstream in a pathway that leads to TRPV channel activation. © 2022 Society of Chemical Industry.

Selective actions of insecticides on desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach (Periplaneta americana) neurons.

BACKGROUND: Insect desensitizing nicotinic acetylcholine (nAChD) receptors are desensitized by low concentrations of agonists, including neonicotinoid insecticides, but are essentially insensitive to spinosyns, while non-desensitizing nicotinic acetylcholine (nAChN) receptors are selectively activated by spinosyns and relatively insensitive to neonicotinoids. RESULTS: The single-electrode voltage-clamp technique was used to measure the actions of newer nicotinic insecticides dinotefuran, sulfoxaflor, triflumezopyrim, spinetoram and GS-ω/k-hexatoxin-Hv1a on cockroach neuronal nAChD and nAChN currents. Like imidacloprid and clothianidin, newer orthosteric nicotinic agonist insecticides dinotefuran and sulfoxaflor act by desensitizing nAChD receptors. The mesoionic insecticide triflumezopyrim selectively inhibited nAChD current with an half maximal inhibitory concentration (IC50 ) of 1.2 nmol L-1 , with no activation. Unlike other Group 4 insecticides, it did not activate nAChN current, but inhibited it with an IC50 of 3.8 µmol L-1 , indicating that the compound is a true antagonist. Spinosad and the spinosyn-derived insecticide spinetoram potently and selectively activated nAChN receptors. GS-ω/k-hexatoxin-Hv1a had no effect on nAChN currents and it had a complex action on nAChD currents, inhibiting at sub-nanomolar concentrations and causing some activation and enhancement of ACh-evoked currents at 30 nmol L-1 and above. Some cells express GS-ω/k-hexatoxin-Hv1a-resistant nAChD receptors. CONCLUSIONS: Nicotinic acetylcholine receptor competitive modulators (IRAC Group 4) and nicotinic acetylcholine receptor allosteric modulators, site II (hexatoxins, IRAC Group 32) are selective for nAChD receptors, while nicotinic acetylcholine receptor allosteric modulators, site I (spinosyns, IRAC Group 5) are selective for nAChN receptors. It is proposed that IRAC Groups 5 and 32 be re-named non-desensitizing nicotinic acetylcholine receptor allosteric modulators and desensitizing nicotinic acetylcholine receptor allosteric modulators, respectively. © 2021 Society of Chemical Industry.

Cholinergic currents in leg motoneurons of Carausius morosus.

We used patch-clamp recordings and fast optical Ca(2+) imaging to characterize an acetylcholine-induced current (I(ACh)) in leg motoneurons of the stick insect Carausius morosus. Our long-term goal is to better understand the synaptic and integrative properties of the leg sensory-motor system, which has served extremely successfully as a model to study basic principles of walking and locomotion on the network level. The experiments were performed under biophysically controlled conditions on freshly dissociated leg motoneurons to avoid secondary effects from the network. To allow for unequivocal identification, the leg motoneurons were backfilled with a fluorescent label through the main leg nerve prior to cell dissociation. In 87% of the motoneurons, I(ACh) consisted of a fast-desensitizing (I(ACh1)) and a slow-desensitizing component (I(ACh2)), both of which were concentration dependent, with EC(50) values of 3.7 x 10(-5) and 2.0 x 10(-5) M, respectively. Ca(2+) imaging revealed that a considerable portion of I(ACh) ( approximately 18%) is carried by Ca(2+), suggesting that I(ACh), besides mediating fast synaptic transmission, could also induce Ca(2+)-dependent processes. Using specific nicotinic and muscarinic acetylcholine receptor ligands, we showed that I(ACh) was exclusively mediated by nicotinic acetylcholine receptors. Distinct concentration-response relations of I(ACh1) and I(ACh2) for these ligands indicated that they are mediated by different types of nicotinic acetylcholine receptors.

Mechanism of action of sodium channel blocker insecticides (SCBIs) on insect sodium channels.

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides, with a mechanism of action different from those of other classes of insecticides that target voltage-gated sodium channels. These compounds have no effect at hyperpolarized membrane potentials, but cause a voltage-dependent, nearly irreversible block as the membrane potential is depolarized. The mechanism of action of SCBIs is similar to that of local anesthetics (LAs), class I anticonvulsants and class I antiarrhythmics. In this article, we review the physiological actions of these compounds on the whole animal, the nervous system and sodium channels, and also present the results from recent studies that elucidate the receptor site of SCBIs.

Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals.

Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABA(A) receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC(50)s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABA(A) receptors. Moreover, GluCls of cockroach neurons had low IC(50)s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC(50)s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.

Ticks home in on body heat: A new understanding of Haller's organ and repellent action.

Ticks are second only to mosquitoes as vectors of disease to humans and animals. Tick host detection is mainly ascribed to Haller's organ, a complex sensory structure on the tick foreleg that detects odors, carbon dioxide and heat, but these host detection mechanisms are not well understood. There is anecdotal evidence that ticks and other ectoparasites are attracted to heat, but it has never been demonstrated that they use radiant heat to detect hosts at a distance. In fact, previous attempts to do this have concluded that radiant heat was not used by ticks. Here we use a novel thermotaxis assay to investigate the detection range, temperature dependence and repellent sensitivity of heat perception in ticks and to identify the sensory organ responsible for this sense. We show that Amblyomma americanum and Dermacentor variabilis ticks can locate a human from several meters away by radiant heat sensed by the part of Haller's organ known as the capsule, a covered spherical pit organ. An aperture in the capsule cover confers directionality and highly reflective interior surfaces of the capsule concentrate radiation on the sensilla to sharpen directionality and increase sensitivity. Commercial insect repellents provide an effective means of personal protection against potentially infectious tick bites by hindering host-seeking behavior. Low concentrations of the insect repellents DEET, picaridin, 2-undecanone, citronellal and nootkatone eliminate thermotaxis without affecting olfaction-stimulated host-seeking behavior. Our results demonstrate that the tick Haller's organ capsule is a radiant heat sensor used in host-finding and that repellents disrupt this sense at concentrations that do not disrupt olfaction. We anticipate that this discovery will significantly aid insect repellent research and provide novel targets for the development of innovative integrated pest management programs and personal protection strategies for ectoparasites and vector-borne disease.

Neural actions of imidacloprid and their involvement in resistance in the Colorado potato beetle, Leptinotarsa decemlineata (Say).

BACKGROUND: The development of resistance to imidacloprid in eastern US populations of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), threatens this critical use for neonicotinoid insecticides. Previous pharmacokinetic studies with resistant adult CPBs provided no explanation for the high resistance level (over 200-fold) to topically applied imidacloprid. The authors assessed the neural activity of imidacloprid by recording spontaneous activity from a motor nerve leaving the isolated central nervous system to compare the sensitivity of the latter to imidacloprid between susceptible and resistant CPBs. RESULTS: On the isolated central nervous system, imidacloprid was initially neuroexcitatory, and neuroinhibitory at higher concentrations. The neuroexcitatory action of imidacloprid was blocked by coapplication of a specific nAChR antagonist, methyllycaconitine, indicating that it is a result of action on nAChRs. The sensitivity to the neuroexcitatory and inhibitory activities of imidacloprid varied independently among individuals in each population. The sensitivity of the central nervous system of resistant CPBs to excitation by imidacloprid did not differ from that of susceptible insects, but the sensitivity to inhibition by imidacloprid was reduced 52- to 58-fold, indicating a possible change in the sensitivity of at least one subgroup of nAChRs. CONCLUSION: This study provides evidence that reduced nerve sensitivity to the blocking action of imidacloprid is associated with imidacloprid resistance in the CPB.

Insect TRP channels as targets for insecticides and repellents.

This review provides a brief overview of ion channels, then focuses on TRP channels, describing the properties and functions of the seven TRP channel classes found in insects. Finally, recent work showing that a heteromeric channel composed of Nanchung and Inactive vanilloid TRP (TRPV) channel subunits is the target of the selective feeding blockers pymetrozine and pyrifluquinazon is described. The possible utility of other TRP channels as targets of insecticides and repellents is also considered.

Chance and design in proinsecticide discovery.

Many insecticides are inactive on their target sites in the form that is sold and applied, needing first to be bioactivated. This proinsecticide strategy has often been achieved by design, through systematic derivatization of intrinsically active molecules with protecting groups that mask their toxic effects until their selective removal in target insects by metabolic enzymes generates the toxiphore. Proinsecticides can be designed to gain selectivity between target and non-target organisms, or to improve bioavailability by enhancing plant or insect uptake. In most cases, however, chance trumps design in proinsecticide discovery: most first-in-class products that we now know to be proinsecticides were only discovered a posteriori to be such, often after having been on the market for years. Knowing the active form of an insecticide is essential to mode of action identification, and early mode of action studies on novel chemotypes should take into account the possibility that the compounds might be proinsecticides. This paper reviews examples of proinsecticides in the marketplace, strategies for making proinsecticides and techniques for unmasking proinsecticides in mode of action studies. Our analysis of global agrochemical sales data shows that 34% of the dollar value of crop insecticides used in 2015 were proinsecticides. © 2016 Society of Chemical Industry.

Afidopyropen: New and potent modulator of insect transient receptor potential channels.

The commercial insecticides pymetrozine and pyrifluquinazon control plant-sucking pests by disturbing their coordination and ability to feed. We have previously shown that these compounds act by overstimulating and eventually silencing vanilloid-type transient receptor potential (TRPV) channels, which consist of two proteins, Nanchung and Inactive, that are co-expressed exclusively in insect chordotonal stretch receptor neurons. Here we show that a new insecticidal compound, afidopyropen, modulates chordotonal organs of American grasshoppers (Schistocerca americana) in the same fashion. Afidopyropen stimulated heterologously expressed TRPV channels from two different insect species - fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum) - but did not affect function of the mammalian TRPV channel TRPV4. Activation of the insect TRPVs required simultaneous expression of both Nanchung and Inactive proteins. Tritium-labeled afidopyropen bound fruit fly TRPVs with higher affinity than pymetrozine and competed with pymetrozine for binding. Nanchung protein formed the main binding interface for afidopyropen, whereas co-expression of Inactive dramatically increased binding affinity. Another modulator of chordotonal organs, flonicamid, did not activate insect TRPV channels, nor did it compete with afidopyropen for binding, indicating that it has a different target site. These results define afidopyropen as a new, potent and specific modulator of insect TRPV channels, and provide insight into the unique binding mode of these compounds.

Antagonist pharmacology of desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach neurons.

Two α-bungarotoxin-sensitive nicotinic acetylcholine (ACh) receptor subtypes in neurons of the American cockroach have been identified as desensitizing (nAChD) and selectively inhibitable with 100nM imidacloprid, and non-desensitizing (nAChN) and selectively inhibitable with 100pM methyllycaconitine. In this paper, the single-electrode voltage-clamp technique was used to measure concentration-response relations for the action of ACh and five antagonists on pharmacologically separated nAChD and nAChN receptors of acutely dissociated neurons from thoracic ganglia of the American cockroach. A dual bath and U-tube perfusion system was used to achieve rapid application of ACh in the continued presence of antagonists, which was essential to accurately measure inhibition by rapidly-reversible antagonists. ACh activated both receptors with an EC50 of 7µM and the antagonist potencies were (nAChD/nAChN in nM): dihydro-ß-erythroidine: 1.0/5.6, d-tubocurarine: 1000/34, condelphine: 0.39/0.65, phencyclidine: 74/980 and mecamylamine 47/1150. While each of these antagonists displayed some subtype selectivity, none are selective enough to be used as subtype-selective tools. These results bring to a total of 16 the number of nicotinic compounds that have been measured on nAChD and nAChN currents. Characterization of these receptors is important for understanding the role of nAChRs in the insect nervous system and the mechanism of action of insecticides.

Block of two subtypes of sodium channels in cockroach neurons by indoxacarb insecticides.

Indoxacarb, a novel insecticide, and its decarbomethoxyllated metabolite, DCJW, are known to block voltage-gated Na(+) channels in insects and mammals, but the mechanism of block is not yet well understood. The present study was undertaken to characterize the action of indoxacarb and DCJW on cockroach Na(+) channels. Na(+) currents were recorded using the whole-cell patch clamp technique from neurons acutely dissociated from thoracic ganglia of the American cockroach Periplaneta americana L. Two types of tetrodotoxin-sensitive Na(+) currents were observed, with different voltage dependencies of channel inactivation. Type-I Na(+) currents were inactivated at more negative potentials than type-II Na(+) currents. As a result, these two types of Na(+) channels responded to indoxacarb compounds differentially. At a holding potential of -100 mV, type-I Na(+) currents were inhibited reversibly by 1 microM indoxacarb and irreversibly by 1 microM DCJW in a voltage-dependent manner, whereas type-II Na(+) currents were not affected by either of the compound. However, type-II Na(+) currents were inhibited by indoxacarb or DCJW at more depolarizing membrane potentials, ranging from -60 to -40 mV. The slow inactivation curves of type-I and type-II Na(+) channels were significantly shifted in the hyperpolarizing direction by indoxacarb and DCJW, suggesting that these compounds have high affinities for the inactivated state of the Na(+) channels. It was concluded that the differential blocking actions of indoxacarb insecticides on type-I and type-II Na(+) currents resulted from their different voltage dependence of Na(+) channel inactivation. The irreversible nature of DCJW block may be partially responsible for its potent action in insects.

TRP Channels in Insect Stretch Receptors as Insecticide Targets.

Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

Kinetic and pharmacological characterization of desensitizing and non-desensitizing glutamate-gated chloride channels in cockroach neurons.

Glutamate-gated chloride channels (GluCls) are found only in invertebrate nerve and muscle, where they mediate inhibitory synaptic transmission, and are important target sites of insecticides. Two GluCl subtypes have previously been distinguished in isolated cockroach CNS neurons based on differential pharmacology. The present study characterizes the kinetics and pharmacological properties of desensitizing and non-desensitizing GluCls. Both types of GluCls were sensitive to glutamate and ibotenic acid. The non-desensitizing GluCl subtype was elicited by glutamate with an EC(50) of 115.8 microM and a Hill coefficient of 2.6 and was also sensitive to the agonist ibotenic acid with an EC(50) of 42 microM and a Hill coefficient of 1.7. The desensitizing and non-desensitizing currents were carried by chloride ions, and occurred either separately or in combination in individual neurons. The GluCls were also found to coexist with and function independently of the GABA-activated chloride channels. The desensitizing and non-desensitizing GluCls exhibited different sensitivities to the ligand-gated channel blocker picrotoxinin. The desensitizing GluCls were blocked only 8% by 30 microM picrotoxinin, whereas the non-desensitizing GluCls were potently blocked by picrotoxinin with an IC(50) of 4.1 microM. The insecticides fipronil and dieldrin at 1 microM inhibited the desensitizing currents by 56 and 13%, respectively, and the non-desensitizing currents by 98 and 43%, respectively. It is concluded that the two types of GluCls found in cockroach neurons exhibit significantly different electrophysiological and pharmacological characteristics.

Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons.

Two alpha-bungarotoxin-sensitive nicotinic receptor subtypes in cockroach neurons are identified as desensitizing (nAChD), selectively inhibitable with 100 nM imidacloprid, and non-desensitizing (nAChN), selectively inhibitable with 100 pM methyllycaconitine. Although the desensitization rate of nAChD receptors is highly variable, pharmacology is largely independent of desensitization rate. Because desensitized states tightly bind agonists, nAChD receptors are potently inhibited by neonicotinoids and specifically measured in radiolabeled imidacloprid binding assays. However, they are not usually detected in binding assays with radiolabeled alpha-bungarotoxin, which has a Kd for the resting state of 21 nM, but binds poorly to desensitized states often present in binding assays. In contrast, nAChN receptors are specifically measured in binding assays with radiolabeled alpha-bungarotoxin, which binds them with a Kd of 1.3 nM. nAChN receptors are activated by neonicotinoids at micromolar concentrations, and allosterically by spinosyn A, with an EC50 of 27 nM. Spinosyn A weakly antagonizes nAChD receptors -23% at 10 microM. The roles of the two nAChR subtypes in insecticide poisoning are discussed.

Voltage-Gated Sodium Channels as Insecticide Targets.

Voltage-gated sodium channels are critical for the generation and propagation of action potentials. They are the primary target of several classes of insecticides, including DDT, pyrethroids and sodium channel blocker insecticides (SCBIs). DDT and pyrethroids preferably bind to open sodium channels and stabilize the open state, causing prolonged currents. In contrast, SCBIs block sodium channels by binding to the inactivated state. Many sodium channel mutations are associated with knockdown resistance (kdr) to DDT and pyrethroids in diverse arthropod pests. Functional characterization of kdr mutations together with computational modelling predicts dual pyrethroid receptor sites on sodium channels. In contrast, the molecular determinants of the SCBI receptor site remain largely unknown. In this review, we summarize current knowledge about the molecular mechanisms of action of pyrethroids and SCBIs, and highlight the differences in the molecular interaction of these insecticides with insect versus mammalian sodium channels.