Histrionicotoxin (HTX) alkaloids, which are isolated from Colombian poison dart frogs, are analgesic neurotoxins that modulate nicotinic acetylcholine receptors (nAChRs) as antagonists. Perhydrohistrionicotoxin (pHTX) is the potent synthetic analogue of HTX and possesses a 1-azaspiro[5.5]undecane skeleton common to the HTX family. Here, we show for the first time the divergent nine-step synthesis of pHTX and its three stereoisomers from the known aldehyde through a one-step construction of the 1-azaspiro[5.5]undecane framework from a linear amino ynone substrate. Surprisingly, some pHTX diastereomers exhibited antagonistic activities on the chicken α4β2-neuronal nAChRs that were more potent than pHTX.

Researchers have established that (+)-7-iso-jasmonic acid ((+)-7-iso-JA) is an intermediate in the production of cis-jasmone (CJ); however, the biosynthetic pathway of CJ has not been fully described. Previous reports stated that CJ, a substructure of pyrethrin II produced by pyrethrum (Tanacetum cinerariifolium), is not biosynthesized through this biosynthetic pathway. To clarify the ambiguity, stable isotope-labelled jasmonates were synthesized, and compounds were applied to apple mint (Mentha suaveolens) via air propagation. The results showed that cis-jasmone is not generated from intermediate (+)-7-iso-JA, and (+)-7-iso-JA is not produced from 3,7-dideydro-JA (3,7-ddh-JA); however, 3,7-didehydro-JA and 4,5-didehydro-7-iso-JA were converted into CJ and JA, respectively.

Decalin-containing tetramic acid is a bioactive scaffold primarily produced by filamentous fungi. The structural diversity of this group of compounds is generated by characteristic enzymes of fungal biosynthetic pathways, including polyketide synthase/nonribosomal peptide synthetase hybrid enzymes and decalin synthase, which are responsible for the construction of a linear polyenoyl tetramic acid structure and stereoselective decalin formation via the intramolecular Diels-Alder reaction, respectively. Compounds that differed only in the decalin configuration were collected from genetically engineered mutants derived from decalin-containing tetramic acid-producing fungi and used for a structure-activity relationship study. Our evaluation of biological activities, such as cytotoxicity against several cancer cell lines and antibacterial, antifungal, antimalarial, and mitochondrial inhibitory activities, demonstrated that the activity for each assay varies depending on the decalin configurations. In addition to these known biological activities, we revealed that the compounds showed inhibitory activity against the insect steroidogenic glutathione S-transferase Noppera-bo. Engineering the decalin configurations would be useful not only to find derivatives with better biological activities but also to discover overlooked biological activities.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs) and their adverse effects on non-target insects are of serious concern. We recently found that cofactor TMX3 enables robust functional expression of insect nAChRs in Xenopus laevis oocytes and showed that neonicotinoids (imidacloprid, thiacloprid, and clothianidin) exhibited agonist actions on some nAChRs of the fruit fly (Drosophila melanogaster), honeybee (Apis mellifera) and bumblebee (Bombus terrestris) with more potent actions on the pollinator nAChRs. However, other subunits from the nAChR family remain to be explored. We show that the Dα3 subunit co-exists with Dα1, Dα2, Dβ1, and Dβ2 subunits in the same neurons of adult D. melanogaster, thereby expanding the possible nAChR subtypes in these cells alone from 4 to 12. The presence of Dα1 and Dα2 subunits reduced the affinity of imidacloprid, thiacloprid, and clothianidin for nAChRs expressed in Xenopus laevis oocytes, whereas the Dα3 subunit enhanced it. RNAi targeting Dα1, Dα2 or Dα3 in adults reduced expression of targeted subunits but commonly enhanced Dβ3 expression. Also, Dα1 RNAi enhanced Dα7 expression, Dα2 RNAi reduced Dα1, Dα6, and Dα7 expression and Dα3 RNAi reduced Dα1 expression while enhancing Dα2 expression, respectively. In most cases, RNAi treatment of either Dα1 or Dα2 reduced neonicotinoid toxicity in larvae, but Dα2 RNAi enhanced neonicotinoid sensitivity in adults reflecting the affinity-reducing effect of Dα2. Substituting each of Dα1, Dα2, and Dα3 subunits by Dα4 or Dβ3 subunit mostly increased neonicotinoid affinity and reduced efficacy. These results are important because they indicate that neonicotinoid actions involve the integrated activity of multiple nAChR subunit combinations and counsel caution in interpreting neonicotinoid actions simply in terms of toxicity.

Natural pesticides pyrethrins biosynthesized by Tanacetum cinrerariifolium are biodegradable and safer insecticides for pest insect control. TcGLIP, a GDSL lipase underpinning the ester bond formation in pyrethrins, exhibits high stereo-specificity for acyl-CoA and alcohol substrates. However, it is unknown how the enzyme recognizes the other structural features of the substrates and whether such specificity affects the product amount and composition in T. cinrerariifolium. We report here that the cysteamine moiety in (1R,3R)-chrysanthemoyl CoA and the conjugated diene moiety in (S)-pyrethrolone play key roles in the interactions with TcGLIP. CoA released from chrysanthemoyl CoA in the pyrethrin-forming reaction reduces the substrate affinity for TcGLIP by feedback inhibition. (S)-Pyrethrolone shows the highest catalytic efficiency for TcGLIP, followed by (S)-cinerolone and (S)-jasmololone, contributing, at least in part, to determine the pyrethrin compositions in T. cinerariifolium.

Meroterpenoid compounds chrodrimanins produced by Talaromyces sp. YO-2 have been shown to act as competitive antagonists of silkworm larval GABAA receptors using electrophysiology, yet no further evidence has been provided to support such an action. We have investigated the actions of chrodrimanin B on rat brain GABAA receptors by binding assays with non-competitive ligand of GABAA receptors [3H]EBOB and competitive ligands [3H]gabazine and [3H]muscimol. Chrodrimanin B did not significantly affect the binding of [3H]EBOB while reducing the binding of [3H]gabazine and [3H]muscimol to the rat membrane preparations. Chrodrimanin B increased the dissociation constant Kd of [3H]gabazine and [3H]muscimol without significantly affecting the maximum binding, pointing to competitive interactions of chrodrimanin B with rat GABAA receptors in support of our previous observation that the compound acts as a competitive antagonist on the silkworm larval GABA receptor.

Tanacetum cinerariifolium was known to produce pyrethrins, but the mechanism of pyrethrin biosynthesis was largely unclear. The author showed that the non-mevalonate and oxylipin pathways underlie biosynthesis of the acid and alcohol moieties, respectively, and a GDSL lipase joins the products of these pathways. A blend of the green leaf volatiles and (E)-β-farnesene mediates the induction of wounding responses to neighboring intact conspecies by enhancing pyrethrin biosynthesis. Plants fight against herbivores underground as well as aboveground, and, in soy pulps, some fungi produce compounds selectively modulating ion channels in insect nervous system. The author proposed that indirect defense of plants occurs where microorganisms produce defense substances in the rhizosphere. Broad-spectrum pesticides, including neonicotinoids, may affect non-target organisms. The author discovered co-factors enabling functional expression of insect nicotinic acetylcholine receptors (nAChRs). This led to understanding the mechanism of insect nAChR-neonicotinoid interactions, thus paving new avenues for controlling pests and disease vectors.

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.

Pyrethrum extracts from flower heads of Chrysanthemum spp. have been used worldwide in insecticides and repellents. While the molecular mechanisms of its insecticidal action are known, the molecular basis of pyrethrum repellency remains a mystery. In this study, we find that the principal components of pyrethrum, pyrethrins, and a minor component, (E)-β-farnesene (EBF), each activate a specific type of olfactory receptor neurons in Aedes aegypti mosquitoes. We identify Ae. aegypti odorant receptor 31 (AaOr31) as a cognate Or for EBF and find that Or31-mediated repellency is significantly synergized by pyrethrin-induced activation of voltage-gated sodium channels. Thus, pyrethrum exerts spatial repellency through a novel, dual-target mechanism. Elucidation of this two-target mechanism may have potential implications in the design and development of a new generation of synthetic repellents against major mosquito vectors of infectious diseases.

The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.

The difficulty of achieving robust functional expression of insect nicotinic acetylcholine receptors (nAChRs) has hampered our understanding of these important molecular targets of globally deployed neonicotinoid insecticides at a time when concerns have grown regarding the toxicity of this chemotype to insect pollinators. We show that thioredoxin-related transmembrane protein 3 (TMX3) is essential to enable robust expression in Xenopus laevis oocytes of honeybee (Apis mellifera) and bumblebee (Bombus terrestris) as well as fruit fly (Drosophila melanogaster) nAChR heteromers targeted by neonicotinoids and not hitherto robustly expressed. This has enabled the characterization of picomolar target site actions of neonicotinoids, findings important in understanding their toxicity.

Neonicotinoids targeting insect nicotinic acetylcholine (ACh) receptors (insect nAChRs) are used for crop protection, but there is a concern about adverse effects on pollinators such as honeybees (Apis mellifera). Thus, we investigated the agonist actions of neonicotinoids (imidacloprid, thiacloprid and clothianidin) on A. mellifera α1 (Amα1)/chicken β2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(β2)2 and (Amα1)2(β2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(β2)2 and (Amα1)2(β2)3 nAChRs with similar current amplitude. We investigated the agonist activity of imidacloprid, thiacloprid and clothianidin for the two hybrid nAChRs and found that: 1) imidacloprid showed higher affinity than clothianidin, whereas clothianidin showed higher efficacy than imidacloprid for the nAChRs; 2) Thiacloprid showed the highest agonist affinity and the lowest efficacy for the nAChRs. The Amα1/β2 subunit ratio influenced the efficacy of imidacloprid and thiacloprid, but hardly affected that of clothianidin. Hydrogen bond formation by the NH group in clothianidin with the main chain carbonyl of the loop B may account, at least in part, for the unique agonist actions of clothianidin on the hybrid nAChRs tested.

Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit.

Pyrethrum (Tanacetum cinerariifolium) produces insecticidal compounds known as pyrethrins. Pyrethrins are esters; the acid moiety is either trans-chrysanthemic acid or pyrethric acid and the alcohol moiety of pyrethrins is either pyrethrolone, cinerolone, or jasmolone. It was generally accepted that cis-jasmone was biosynthetic intermediate to produce the alcohol moieties of pyrethrin, and the biosynthetic origin of the cis-jasmone was postulated to be jasmonic acid. However, there was no direct evidence to prove this hypothesis. In order to uncover the origin of pyrethrolone moiety in pyrethrin II, feeding experiments were performed employing deuterium- and 13C-labeled compounds as substrates, and the expected labeled compounds were analyzed using UPLC MS/MS system. It was found that the pyrethrolone moiety in pyrethrin II was derived from 12-oxo-phytodienoic acid (OPDA), iso-OPDA and cis-jasmone but not from methyl jasmonate and 3-oxo-2-(2'-[Z]-pentenyl)-cyclopentane-1-hexanoic acid. The results supported that the biosynthesis of the pyrethrolone moiety in pyrethrin II partially used part of the jasmonic acid biosynthetic pathway, but not whole.

© Copyright © 2020 Sakurai, Mardani-Korrani, Nakayasu, Matsuda, Ochiai, Kobayashi, Tahara, Onodera, Aoki, Motobayashi, Komatsuzaki, Ihara, Shibata, Fujii and Sugiyama. Inter-organismal communications below ground, such as plant–microbe interactions in the rhizosphere, affect plant growth. Metabolites are shown to play important roles in biological communication, but there still remain a large number of metabolites in soil to be uncovered. Metabolomics, a technique for the comprehensive analysis of metabolites in samples, may uncover the molecules that intermediate these interactions. We conducted a multivariate analysis using liquid chromatography (LC)—mass spectrometry (MS)-based untargeted metabolomics in several soil samples and also targeted metabolome analysis for the identification of the candidate compounds in soil. We identified okaramine A, B, and C in the rhizosphere soil of hairy vetch. Okaramines are indole alkaloids first identified in soybean pulp (okara) inoculated with Penicillium simplicissimum AK-40 and are insecticidal. Okaramine B was detected in the rhizosphere from an open field growing hairy vetch. Okaramine B was also detected in both bulk and rhizosphere soils of soybean grown following hairy vetch, but not detected in soils of soybean without hairy vetch growth. These results suggested that okaramines might be involved in indirect defense of plants against insects. To our knowledge, this is the first report of okaramines in the natural environment. Untargeted and targeted metabolomics would be useful to uncover the chemistry of the rhizosphere.

Copyright © 2020 by Annual Reviews. All rights reserved. Neonicotinoids have been used to protect crops and animals from insect pests since the 1990s, but there are concerns regarding their adverse effects on nontarget organisms, notably on bees. Enhanced resistance to neonicotinoids in pests is becoming well documented. We address the current understanding of neonicotinoid target site interactions, selectivity, and metabolism not only in pests but also in beneficial insects such as bees. The findings are relevant to the management of both neonicotinoids and the new generation of pesticides targeting insect nicotinic acetylcholine receptors.

In our previous report, it was found that Lasiodiplodia theobromae produced cis-jasmone via partially utilizing the biosynthetic pathway of JA. A feeding experiment using uniformly 13C-labeled α-linolenic acid, which was added to the culture media of the fungus, strongly supported that the fungus produced CJ via the decarboxylation step of the biosynthetic pathway.

© 2018 Elsevier Inc. Neonicotinoids are insecticides that target insect nicotinic acetylcholine receptors (nAChRs), exhibiting high selective toxicity to insects over vertebrates and good systemic activity in crop plants. For these reasons, neonicotinoids currently make up ∼30% of insecticide sales worldwide. However, due to their adverse impact on pollinators such as honey bees and bumble bees, neonicotinoids are being banned from the EU, and other countries may follow. It is therefore crucial to understand the mechanism underlying neonicotinoid actions on pollinators as well as on the nAChRs of pests, with a view to understanding their selectivity. Here we review the molecular mechanisms of neonicotinoid actions at an atomic level, through structural and resistance mechanism studies and propose relevant research topics for further studies on the future of pest management.

© 2018 Elsevier Inc. Neonicotinoid insecticides interact with the orthosteric sites of nicotinic acetylcholine receptors (nAChRs) formed at the interfaces of (a) two adjacent α subunits and (b) α and non-α subunits. However, little is known of the detailed contributions of these two orthosteric sites to neonicotinoid actions. We therefore applied voltage-clamp electrophysiology to the Dα1/chicken β2 hybrid nAChR expressed in Xenopus laevis oocytes to explore the agonist actions of imidacloprid and thiacloprid on wild type receptors and following binding site mutations. First, we studied the S221E mutation in loop C of the ACh binding site of the Dα1 subunit. Secondly, we explored the impact of combining this mutation in loop C with others in the loop D-E-G triangle (R57S; E78K; K140T; S221E). The S221E loop C mutation alone reduced the affinity of the neonicotinoids tested, while hardly affecting the concentration-response curve for acetylcholine. Addition of the three R57S; E78K; K140T mutations in the loop D-E-G triangle led to a further reduction in neonicotinoid sensitivity, suggesting that all four binding site loops (C, D, E, G) in the Dα1 subunit, which are located upstream of loop B in the N-terminal, extracellular domain, contribute to the selective actions of neonicotinoid insecticides.

© 2018 Elsevier Inc. Pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I and jasmolin II are six closely related insecticidal active esters, known as pyrethrins, found in the pyrethrum extract from the dry flowers of Tanacetum cinerariifolium. The chemical structures of the six compounds differ only in the terminal moieties at the acid and alcohol ends, but the compounds’ in vivo toxicities are substantially different. Pyrethrins are lead compounds for pyrethroids, a large family of synthetic insecticides that alter nerve functions by prolonging the opening of voltage-gated sodium channels. However, data on the mechanism of action of natural pyrethrins are very limited. In this study, we examined the actions of all six pyrethrins on cockroach sodium channels expressed in Xenopus oocytes. Although the six compounds showed comparable potencies in inhibiting the inactivation of sodium channels, they had greatly variable potencies in inhibiting channel deactivation. Furthermore, unlike pyrethroids, the action of pyrethrins neither depend on nor were enhanced by repeated channel activation. We created a NavMs-based model of the cockroach sodium channel, in which pyrethrin II was docked at the pyrethroid receptor site 1 (PyR1), and proposed a rationale for the observed structure-activity relationship of the six pyrethrins. Our study sheds light on the molecular mechanism of pyrethrum action on sodium channels and reveled differences in the modes of action of the six bioactive constitutes of pyrethrum.

© 2018 A novel L-glutamate-gated anion channel (IscaGluCl1) has been cloned from the black-legged tick, Ixodes scapularis, which transmits multiple pathogens including the agents of Lyme disease and human granulocytic anaplasmosis. When mRNA encoding IscaGluCl1 was expressed in Xenopus laevis oocytes, we detected robust 50–400 nA currents in response to 100 μM L-glutamate. Responses to L-glutamate were concentration-dependent (pEC50 3.64 ± 0.11). Ibotenate was a partial agonist on IscaGluCl1. We detected no response to 100 μM aspartate, quisqualate, kainate, AMPA or NMDA. Ivermectin at 1 μM activated IscaGluCl1, whereas picrotoxinin (pIC50 6.20 ± 0.04) and the phenylpyrazole fipronil (pIC50 6.90 ± 0.04) showed concentration-dependent block of the L-glutamate response. The indole alkaloid okaramine B, isolated from fermentation products of Penicillium simplicissimum (strain AK40) grown on okara pulp, activated IscaGluCl1 in a concentration-dependent manner (pEC50 5.43 ± 0.43) and may serve as a candidate lead compound for the development of new acaricides.

© 2017 The British Pharmacological Society Background and Purpose: Neonicotinoid insecticides interact with the orthosteric site formed at subunit interfaces of insect nicotinic ACh (nACh) receptors. However, their interactions with the orthosteric sites at α–non α and α–α subunit interfaces remain poorly understood. The aim of this study was to elucidate the mechanism of neonicotinoid actions using the Drosophila Dα1-chicken β2 hybrid nACh receptor. Experimental Approach: Computer models of the (Dα1) 3 (β2) 2 nACh receptor in complex with imidacloprid and thiacloprid were generated. Amino acids in the Dα1 subunit were mutated to corresponding amino acids in the human α4 subunit to examine their effects on the agonist actions of neonicotinoids on (Dα1) 3 (β2) 2 and (Dα1) 2 (β2) 3 nACh receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology. Key Results: The (Dα1) 3 (β2) 2 nACh receptor models indicated that amino acids in loops D, E and G probably determine the effects of neonicotinoids. The amino acid mutations tested had minimal effects on the EC 50 for ACh. However, the R57S mutation in loop G, although having minimal effect on imidacloprid's actions, reduced the affinity of thiacloprid for the (Dα1) 3 (β2) 2 nACh receptor, while scarcely affecting thiacloprid's action on the (Dα1) 2 (β2) 3 nACh receptor. Both the K140T and the combined R57S;K140T mutations reduced neonicotinoid efficacy but only for the (Dα1) 3 (β2) 2 nACh receptor. Combining the E78K mutation with the R57S;K140T mutations resulted in a selective reduction of thiacloprid's affinity for the (Dα1) 3 (β2) 2 nACh receptor. Conclusions and Implications: These findings suggest that a triangle of residues from loops D, E and G contribute to the selective actions of neonicotinoids on insect-vertebrate hybrid nACh receptors. Linked Articles: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

© 2018 American Chemical Society. Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. To explore the biosynthetic potential of okaramines, gene knockout experiments of an okaramine-producer fungus were performed. The deletion mutants of the oxygenase genes okaB, okaD, okaE, and okaG provided analogues that were unlikely to be accumulated in the normal biosynthetic process of the wild-type strain. Analysis of the structure-activity relationships of okaramines collected from the fungal cultures revealed that 1,4-dihydroazocine and N-aliphatic group attached to the indole were crucial for GluCl-activating activity. This provided insights into further derivatization of the complex structure of okaramines in order to facilitate the development of new insecticides.

The pH-sensitive chloride channels (pHCls) are broadly expressed in insects, but little is known about their physiologic role, diversity, and sensitivity to insecticides acting on relevant chloride channels. Here we have sequenced 50 transcripts of the pHCl- 1 gene from the brain, third thoracic ganglion ( T3G), and midgut of larvae of silkworm Bombyx mori. It was found that >50 variants were expressed with distinct splicing in the T3G compared with the brain and midgut. Of the variants detected, variant 9, which was expressed most abundantly in the larvae, was reconstituted in Xenopus laevis oocytes to characterize its pH and ivermectin sensitivity. Variant 9 formed a functional pHCl with half- maximal activation at a pH of 7.87,and was activated by ivermectin irrespective of the extracellular pH. This was in contrast to variant 1, which was activated more profoundly at acidic rather than basic pH. To identify a key determinant for such differential ivermectin sensitivity, different amino acids in variants 1 and 9 were swapped, and the effects of the mutations on ivermectin sensitivity were investigated. The V275S mutation of variant 1 enhanced ivermectin sensitivity, whereas the S275V mutation of variant 9 caused a reduction in sensitivity. In homology models of the Bombyx pHCls, Val275 of variant 1 interacted more strongly with Ala273 than Ser275 of variant 9 at the channel gate. This interaction is likely to prevent ivermectin-induced opening of the channel, accounting, at least partially, for the differential macrolide action on the two variants.

A novel chemotype insecticide flupyrimin (FLP) [N-RE)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloptid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [H-3]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [H-3]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat alpha 4 beta 2 nAChR Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.

In plants, cis-jasmone (CJ) is synthesized from a-linolenic acid (LA) via two biosynthetic pathways using jasmonic acid (JA) and iso-12-oxo-phytodienoic acid (iso-OPDA) as key intermediates. However, there have been no reports documenting CJ production by microorganisms. In the present study, the production of fungal-derived CJ by Lasiodiplodia theobromae was observed for the first time, although this production was not observed for Botrytis cinerea, Verticillium longisporum, Fusarium oxysporum, Gibberella fujikuroi, and Cochliobolus heterostrophus. To investigate the biosynthetic pathway of CJ in L. theobromae, administration experiments using [18,18,18-H-2(3), 17,17-H-2(2)] LA (LA-d5), [18,18,18-H-2(3), 17,17-H-2(2)]12-oxo-phytodienoic acid (cis-OPDA-d5), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 8:0 (OPC8-d6), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 6:0 (OPC6-d6), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 4:0 (OPC4-d6), and [11,11-H-2(2), 10,10-H-2(2), 8,8-H-2(2), 2,2-H-2(2)] methyl iso-12-oxo-phytodienoate (iso-MeOPDA-d8) were carried out, revealing that the fungus produced CJ through a single biosynthetic pathway via iso-OPDA. Interestingly, it was suggested that the previously predicted decarboxylation step of 3,7-didehydroJA to afford CJ might not be involved in CJ biosynthesis in L. theobromae.

The okaramine indole alkaloids were recently shown to be more selective than ivermectin in activating the glutamate-gated chloride channels of the silkworm larvae of Bombyx mori (BmGluCls). Those studies were carried out using the exon 3b variant as a representative of BmGluCls. However, it remains unknown whether okaramines are similarly effective on other silkworm GluCl variants and whether they share the same binding site as ivermectin on GluCls. To begin to address these questions, we examined the potency of four okaramines on the exon 3c variant of BmGluCls by two-electrode voltage clamp voltage recordings of glutamate-induced chloride currents. The potency of okaramines in activating the exon 3c BmGluCI agreed well with findings on the exon 3b BmGluCl and insecticidal potency. Okaramine B (10 mu M) reduced the maximum binding (B-max) but not the dissociation constant (K-D) of [H-3]ivermectin in studies on plasma membrane fractions of HEK293 cells expressing the exon 3c variant. These findings indicate that activation of GluCls is important in the insecticidal actions of okaramines. (C) 2016 Elsevier B.V. All rights reserved.

Although some fungi produce mycotoxins that render food on which they grow inedible, other fungi produce metabolites that, in addition, to human and animal healthcare, are useful in pest control. Penicillium simplicissimum AK-40 produces okaramines as an insecticide when grown on okara, a by-product of soybean curd production. Okaramines selectively activate glutamate-gated chloride channels expressed only in the nervous system of invertebrates. Asperparalines from Aspergillus japonicus JV-23 and chrodrimanins from Talaromyces sp. YO-2, both of which were isolated similarly to okaramines, selectively block insect nicotinic acetylcholine and γ-aminobutyric acid receptors, respectively. These results suggest that fungi induced by certain plant factors have potential for producing next-generation pesticide leads.

GABA receptor Cl- ion channel complex (GABA-gated chloride channel) is a scientifically and practically important target site for various pharmaceutical and animal health drugs, and insecticides. gamma-Benzene hexachloride (gamma-BHC, lindane, one of eight BHC isomers) is well known to be a noncompetitive antagonist of the GABA-gated chloride channel. However why only gamma-BHC, out of eight BHC isomers is insecticidal, remains unclear. In the early 1970s a number of gamma-BHC analogs were synthesized, in which some chlorine atoms were replaced by other substitutes such as hydrogen, halogens other than chlorine and alkoxy groups, etc. Among these analogs, gamma-BHC was the most insecticidal against the housefly and the German cockroach. Recently, we have focused on the polychlorinated gamma-BHC analogs having four to eight chlorine (Cl) atoms on the cyclohexane ring and studied not only their insecticidal but also GABA antagonist activities to elucidate the molecular requirements and the role of Cl atom(s) for gamma-BHC activity.

Background: Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. Methods: We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. Results: Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. Conclusion: An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research.

Background: gamma-Aminobutyric acid (GABA) receptors play a central role in fast inhibitory neurotransmission in insects. Several classes of insecticides targeting insect GABA-gated chloride channels have been developed. The important resistant to dieldrin GABA receptor subunit (RDL) has been used to investigate insecticide sites of action using radioligands, electrophysiology and site-directed mutagenesis. Although this important subunit readily forms robust functional homomeric receptors when expressed, alternative splicing and RNA A-to-I editing can generate diverse forms of the receptor. Methods: We have reviewed studies on native and recombinant insect GABA-gated chloride channels, their interactions with ligands acting at orthosteric and allosteric sites and their interactions with insecticides. Since some GABA receptor modulators act on L-glutamate-gated chloride channels, some comparisons are included. Results: The actions on GABA-gated chloride channels of polychlorocycloalkanes, cyclodienes, macrocyclic lactones, phenylpyrazoles, isoxazolines, and metadiamides are described and the mechanisms of action of members of these insecticide classes are addressed. Mutations that lead to resistance are discussed as they can be important in developing field diagnostic tests. Toxicity issues relating to insecticides targeting GABA-gated chloride channels are also addressed. Conclusion: An overview of all major insecticide classes targeting insect GABA-gated chloride channels has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native GABA receptors remains unknown and studies to clarify this are needed. Also, the precise sites of action of the recently introduced isoxazolines and meta-diamides will be of interest to pursue.

Okaramines produced by Penicillium simplicissimum AK-40 activate L-glutamate-gated chloride channels (GluCls) and thus paralyze insects. However, the okaramine binding site on insect GluCls is poorly understood. Sequence alignment shows that the equivalent of residue Leucine319 of the okaramine B sensitive Bombyx mori (B. mori) GluCl is a phenylalanine in the okaramine B insensitive B. mori gamma-aminobutyric acid-gated chloride channel of the same species. This residue is located in the third transmembrane (TM3) region, a location which in a nematode GluCl is close to the ivermectin binding site. The B. mori GluCl containing the L319F mutation retained its sensitivity to l-glutamate, but responses to ivermectin were reduced and those to okaramine B were completely blocked.

The pH-sensitive chloride channels (pHCls) are expressed widely in the insect nervous system, but their physiological roles and pesticide sensitivity in Lepidoptera are poorly understood. Here, cDNAs of pHCl variants "A" and "B" were isolated from the head of silkworm (Bombyx mori) larvae and expressed in Xenopus laevis oocytes to characterize their functions and examine their pesticide sensitivity. Variant "A" possesses four entire trans membrane domains (TMs), while variant "B" lacks a part of the TM and the TM3-TM4 linker. Only the A variant formed a chloride channel in oocytes which was activated in response to an increase of pH in the extracellular solution. Neither fipronil nor gamma-benzenehexachloride had a significant blocking effect on the A variant when tested at 10 mu M. By contrast, macrolide ivermectin activated it at acidic pH but blocked it at pH 7.6 at concentrations higher than 300 nM, indicating a likely contribution to in vivo toxicity. (C) 2015 Elsevier Inc. All rights reserved.

© 2016 Taylor & Francis Group, LLC. Natural pyrethrins are used to control household and agricultural pests, and it is of value to understand biosynthesis in Tanacetum cinerariifolium for enhanced production. We previously found that a blend of four green leaf volatiles (GLVs) and (E)-b-farnesene emitted by T. cinerariifolium seedlings enhanced gene expressions of certain biosynthetic enzymes in unwounded seedlings; however, the extent to which such a regulation facilitates pyrethrin biosynthesis remains unknown. Here we have investigated the effects of the blend of the volatile organic compounds (VOCs) on gene expressions of seven biosynthetic enzymes. VOC treatment resulted in enhanced chrysanthemyl diphosphate synthase (CDS), chrysanthemic acid synthase (CAS), Tanacetum cinerariifolium GDSL lipase (TcGLIP) and acyl-Coenzyme A oxidase 1 (ACX1) gene expressions that reached a peak at a 12 h VOC treatment, whereas the treatment minimally influenced the expressions of other biosynthetic genes. In undifferentiated Tanacetum tissues, such VOCinduced amplification of CDS, CAS, TcGLIP and ACX1 gene expressions were markedly reduced, suggesting that a high-resolution, VOC-mediated communication is an event selective to differentiated plants.

© 2015 Elsevier Inc. Neonicotinoid insecticides interact with the orthosteric site on the extracellular ligand binding domain (LBD) of nicotinic acetylcholine receptors (nAChRs), typically activating the cation permeable ion channels. In nAChRs consisting of two α and three non-α subunits, LBDs contain six loops (loops A, B and C on the α subunit and loops D, E and F on the non-α subunit) which make up the orthosteric binding site at the α/non-α subunit interfaces. Recently, an additional site (loop G) on the β1 strand has been identified. Also, when the α/non-α subunit ratio is 3/2, another binding site is generated at the interface of two adjacent α subunits. Roles for loop G and the α-α interface in the interactions with neonicotinoids are discussed with reference to recent structural and physiological data.

Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B-1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode (Haemonchus contortus) and silkworm (Bombyx mori) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B-1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls. (C) 2014 Elsevier Inc. All rights reserved.

The management of weeds and diseases that are caused by phytopathogenic fungi is important for preventing the loss of agricultural products. The aim of the present study was to identify phytotoxic and antiphytopathogenic agents from the Thai Alpinia galanga rhizome. Extracts of the dried rhizomes of A. galanga (Zingiberaceae) were separated and tested for phytotoxic activity against lettuce (Lactuca sativa L. cv. Great Lakes) and Italian ryegrass (Lolium multiflorum Lam. cv. Wasefudou) and for antiphytopathogenic activity against Alternaria porri, Colletotrichum gloeosporioides, Fusarium oxysporum and Phytophthora nicotianae. 1′‐Acetoxychavicol acetate (1) was identified as one of the main components, together with trans‐p‐coumaryl acetate (3) and trans‐p‐acetoxycinnamyl acetate (2). 1′‐Acetoxychavicol acetate (1) was solvolyzed with 2% EtOH to yield trans‐p‐coumaryl ethyl ether (6), trans‐p‐coumaryl acetate (3) and trans‐p‐coumaryl alcohol (5). 1′‐Acetoxychavicol acetate (1) completely inhibited the root growth of the lettuce seedlings at 50 μg mL^–1, but had a weaker inhibitory effect on the growth of Italian ryegrass. 1′‐Acetoxychavicol acetate also inhibited the growth of P. nicotianae and A. porri, with minimum inhibition concentration values of 15.6 and 31.5 μg mL^–1, respectively. The plant growth‐inhibitory activity and fungal growth‐inhibitory activity of trans‐p‐coumaryl acetate (3), trans‐p‐coumaryl ethyl ether, trans‐p‐coumaryl alcohol (5) and trans‐p‐acetoxycinnamyl acetate (2) were lower than those of 1′‐acetoxychavicol acetate. A structure–activity relationship suggested that the strong phytotoxic and antiphytopathogenic activity of 1′‐acetoxychavicol acetate relied on the 1′‐acetoxyl group.

© 2015 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

Glutamate-gated chloride channels (GluCls) mediate fast inhibitory neurotransmission in invertebrate nervous systems. Insect GluCls show alternative splicing, and, to determine its impact on channel function and pharmacology, we isolated GluCl cDNAs from larvae of the silkworm (Bombyx mori). We show that six B. mori glutamate-gated chloride channel variants are generated by splicing in exons 3 and 9 and that exons 3b and 3c are common in the brain and third thoracic ganglion. When expressed in Xenopus laevis oocytes, the three functional exon 3 variants (3a, b, c) all had similar EC50 values for L-glutamate and ivermectin (IVM); however, I-max (the maximum L-glutamate- and IVM-induced response of the channels at saturating concentrations) differed strikingly between variants, with the 3c variant showing the largest L-glutamate- and IVM-induced responses. By contrast, a partial deletion detected in exon 9 had a much smaller impact on L-glutamate and IVM actions. Binding assays using [H-3] IVM indicate that diversity in IVM responses among the GluCl variants is mainly due to the impact on channel assembly, altering receptor cell surface numbers. GluCl variants expressed in HEK293 cells show that structural differences influenced B-max but not K-d values of [H-3] IVM. Domain swapping and site-directed mutagenesis identified four amino acids in exon 3c as hot spots determining the highest amplitude of the L-glutamate and IVM responses. Modeling the GluCl 3a and 3c variants suggested that three of the four amino acids contribute to intersubunit contacts, whereas the other interacts with the TM2-TM3 linker, influencing the receptor response.

Neonicotinoid insecticides target insect nicotinic acetylcholine receptors (nAChRs). Their widespread use and possible risks to pollinators make it extremely urgent to understand the mechanisms underlying their actions on insect nAChRs. We therefore elucidated X-ray crystal structures of the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) and its Gln55Arg mutant, more closely resembling insect nAChRs, in complex with a nitromethylene imidacloprid analog (CH-IMI) and desnitro-imidacloprid metabolite (DN-IMI) as well as commercial neonicotinoids, imidacloprid, clothianidin, and thiacloprid. Unlike imidacloprid, clothianidin, and CH-IMI, thiacloprid did not stack with Tyr185 in the wild-type Ls-AChBP, but did in the Gln55Arg mutant, interacting electrostatically with Arg55. In contrast, DN-IMI lacking the NO2 group was directed away from Lys34 and Arg55 to form hydrogen bonds with Tyr89 in loop A and the main chain carbonyl of Trp143 in loop B. Unexpectedly, we found that several neonicotinoids interacted with Lys34 in loop G on the beta 1 strand in the crystal structure of the Gln55Arg mutant. Basic residues introduced into the alpha 7 nAChR at positions equivalent to AChBP Lys34 and Arg55 enhanced agonist actions of neonicotinoids, while reducing the actions of acetylcholine, (-)-nicotine, and DN-IMI. Thus, not only the basic residues in loop D, but also those in loop G determine the actions of neonicotinoids. These novel findings provide new insights into the modes of action of neonicotinoids and emerging derivatives.

In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) gamma-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 mu M on human alpha 1 beta 2 gamma 2 GABACl and alpha 1 beta glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.

Polymorphisms are sometimes observed in native insect nicotinic acetylcholine receptor (nAChR) subunits, which are important insecticide targets, yet little is known of their impact on insecticide actions. Here we investigated the effects of a polymorphism involving the substitution of histidine108 by leucine in the Drosophila melanogaster D alpha 1 subunit on the agonist actions of the neurotransmitter acetylcholine (ACh) and two commercial neonicotinoid insecticides (imidacloprid and clothianidin). There was no significant impact of the H108L substitution on either the ACh EC50, the concentration leading to a half maximal ACh response, or the maximum current amplitude in response at 10 mu M ACh, of the D alpha 1-chicken beta 2 nAChR expressed in Xenopus laevis oocytes. However, the response amplitudes to imidacloprid and clothianidin were significantly enhanced, indicating a role of His108 in the selective interactions of D alpha 1 with these neonicotinoids.

Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR alpha subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect alpha 1 nAChR group and has been named Rsan alpha 1. Rsan alpha 1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsan alpha 1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken beta 2 nAChR, Rsan alpha 1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 mu M) and choline (100 mu M). Rsan alpha 1/beta 2 was insensitive to both imidacloprid (100 mu M) and spinosad (100 mu M). The unreliable expression of Rsan alpha 1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides. (C) 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

2013 marks the 50th annual Drew festival in Uto City, Japan, celebrating the work of University of Manchester botanist, Dr. Kathleen Drew-Baker. Her insight into the reproductive biology of algae was the key to efficient farming of the seaweed "nori" which is a familiar component of Japanese food. © 2013 WILEY Periodicals, Inc.

Invertebrate gamma-aminobutyric acid (GABA)-gated chloride channels (GABACls) and glutamate-gated chloride channels (GluCls), which function as inhibitory neurotransmitter receptors, are important targets of insecticides and antiparasitic agents. The antagonism of GABACls and GluCls by 4-isobutyl-3-isopropylbicyclophosphorothionate (PS-14) was examined in cultured cockroach and rat neurons using a whole-cell patch-clamp method. The results indicated that PS-14 selectively blocks cockroach GABACls relative to cockroach GluCls and rat GABACls. PS-14 represents a useful probe for the study of insect GABA receptors. (C) 2013 Elsevier Ltd. All rights reserved.

We have recently discovered that a GDSL lipase/ esterase (TcGLIP) in Tanacetum cinerariifolium catalyzed acyltransferase activity to form an ester bond in the natural insecticide, pyrethrin. TcGLIP contained Ser40 in Block I, Gly64 in Block II, Asn168 in Block III and Asp318 and His321 in Block V, suggesting underlying hydrolase activity, although little is known about their role in acyltransferase activity. We expressed TcGLIP here in Esherichia coli as a fusion with maltosebinding protein (MBP), part of the fusion being cleaved with a protease to obtain MBP-free TcGLIP. A kinetic analysis revealed that the MBP moiety scarcely influenced the kinetic parameters. The effects on acyltransferase activity of mutations of Gly64, Asn168, Asp318 and His321 were investigated by using MBP-fused TcGLIP. Mutations of these amino acids markedly reduced the acyltransferase activity, suggesting their critical role in the production of pyrethrins.

A series of 4-(6-imino-3-aryl/heteroarylpyridazin-1-yl) butanoic acids were synthesized and examined for antagonism of GABA receptors from three insect species. When tested against small brown planthopper GABA receptors, the 3,4-methylenedioxyphenyl and the 2-naphthyl analogues showed complete inhibition of GABA-induced fluorescence changes at 100 mu M in assays using a membrane potential probe. Against common cutworm GABA receptors, these analogues displayed approximately 86% and complete inhibition of GABA-induced fluorescence changes at 100 mu M, respectively. The 4-biphenyl and 4-phenoxyphenyl analogues showed moderate inhibition at 10 mu M in these receptors, although the inhibition at 100 mu M was not complete. Against American cockroach GABA receptors, the 4-biphenyl analogue exhibited the greatest inhibition (approximately 92%) of GABA-induced currents, when tested at 500 mu M using a patch-clamp technique. The second most active analogue was the 2-naphthyl analogue with approximately 85% inhibition. The 3-thienyl analogue demonstrated competitive inhibition of cockroach GABA receptors. Homology modeling and ligand docking studies predicted that hydrophobic 3-substituents could interact with an accessory binding site at the orthosteric binding site. (C) 2012 Elsevier Ltd. All rights reserved.

Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of T.similar to cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T.similar to cinerariifolium.

Bifenazate is a very selective acaricide that controls the spider mite, Tetranychus urticae. Bifenazate is the first example of a carbazate acaricide. Its mode of action remains unclear. Bifenazate and its active metabolite diazene induce paralysis in spider mites, suggesting that they may act on the nervous system. Here we have employed a homologue (TuGABAR) of RDL (Resistance to dieldrin), a subunit of ionotropic gamma-aminobutyric acid (GABA) receptor, from T. urticae to investigate the action of bifenazate and its active metabolite diazene on this receptor function. Although neither acaricide showed a GABA agonist action, 30 mu M of bifenazate or diazene significantly enhanced the GABA-induced response of TuGABAR in a dose-dependent manner, shifting the EC50 of GABA from 24.8 mu M to 4.83 mu M and 10.8 mu M, respectively. This action demonstrates a positive allosteric modulator effect of bifenazate on T. urticae GABA receptors. This synergistic action is likely the result of bifenazate binding to a site distinct from that of the GABA binding site causing a conformational change that affects the magnitude of the GABA response. Precisely how the observed GABA synergist action correlates with the acaricidal activity of bifenazate, if at all, has yet to be determined. (c) 2012 Elsevier Inc. All rights reserved.

Glutamate-gated chloride (GluCl) channels are pentameric receptors for the inhibitory neurotransmitter glutamate in invertebrates and are a major target for macrolide anthelmintics. Three amino acids in GluCl channels are reported to render macrolide resistance in nematodes and insects. To examine whether these three amino acids are involved in binding of the antiparasitic agent milbemycin (MLM) to the GluCl channels of the nematode parasite Haemonchus contortus, the equivalent amino acids (L256, P316, and G329) of the Hco-AVR-14B subunit were substituted with various amino acids. cDNAs encoding the wild type and mutants of this subunit were transfected into COS-1 cells for transient expression and analysis of GluCl channels. The abilities of these mutant channels to bind [H-3]MLM A(4) were remarkably decreased when compared with the wild-type channel. In patch clamp analysis, L256F and P3165 mutant channels were 37- and 100-fold less sensitive to MLM A(4) when compared with the wild-type channel, respectively. These findings indicate that amino acid changes in the beta 10 strand, the M2-M3 linker, and the M3 region influence MLM A(4) binding to the channel. Homology modeling and ligand docking studies suggest the presence of two potential binding sites for MLM A(4). (C) 2012 Elsevier Inc. All rights reserved.

Plants emit volatile organic compounds (VOCs) as a means to warn other plants of impending danger. Nearby plants exposed to the induced VOCs prepare their own defense weapons in response. Accumulated data supports this assertion, yet much of the evidence has been obtained in laboratories under artificial conditions where, for example, a single VOC might be applied at a concentration that plants do not actually experience in nature. Experiments conducted outdoors suggest that communication occurs only within a limited distance from the damaged plants. Thus, the question remains as to whether VOCs work as a single component or a specific blend, and at which concentrations VOCs elicit insect and pathogen defenses in undamaged plants. We discuss these issues based on available literature and our recent work, and propose future directions in this field. © 2012 Landes Bioscience.

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Pyrethrins are a natural insecticide biosynthesized by the plant pyrethrum [Chrysanthemum cinerariaefolium (Current species name: Tanacetum cinerariifolium)] of the family Asteraceae. Although pyrethrins have been used to control household pests for the past century, little is known about the mechanism of biosynthesis, contrasting with intensive research on their synthetic analogs, pyrethroids. The author studied pyrethrin biosynthesis in young seedlings of C. cinerariaefolium. The results of experiments using C-13-labeled glucose as the biosynthesis precursor indicated that the acid and alcohol moieties are biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate (MEP) and oxylipin pathways, respectively. Further study on the effects of wound-induced signals in leaves showed that biosynthesis is enhanced in response to both volatile and nonvolatile signals.

Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori) larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 mu M) had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 mu M acetylcholine (ACh), it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR)-selective antaogonist. In contrast, 10 mu M asperparaline A was ineffective on the c-aminobutyric acid-and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 mM ACh in terms of IC50 values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM) reduced the maximum neuron response to ACh with a minimal shift in EC50, suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken alpha 3 beta 4, alpha 4 beta 2 and alpha 7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs.

Plants emit specific blends of volatile organic compounds (VOCs) in response to mechanical wounding. Such induced VOCs have been shown to mediate in plant and interplant communication, yet little is known about the time- and dose-response relationships in VOC-mediated communications. Here, we employed young seedlings of Chrysanthemum cinerariaefolium to examine the effects of volatiles emitted by artificially damaged seedlings on the biosynthesis of the natural insecticides pyrethrins in intact conspecific plants. Wounded leaves emitted (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate and (E)-beta-farnesene as dominant wound-induced VOCs. Exposing intact seedlings to a mixture of these VOCs at concentrations mimicking those emitted from wounded seedlings, as well as placing the intact seedlings next to the wounded seedlings, resulted in enhanced pyrethrin contents in the intact seedlings. Thus we quantified mRNA transcripts of 1-deoxy-d-xylulose 5-phosphate synthase (DXS), chrysanthemyl diphosphate synthase (CPPase), 13-lipoxygenase (13-LOX) and allene oxide synthase (AOS) genes in intact seedlings exposed to the VOC mixture to show that DXS and 13-LOX gene expression reached a maximum at 3 h, whereas CPPase and AOS reached it at 6 h. Interestingly, both increasing and decreasing the VOC mixture concentrations from those observed on injury reduced the expression of DXS, CPPase and AOS genes to the control level. Also, separating the VOC mixture into individual components eliminated the ability to enhance the expression of all the biosynthetic genes examined. This is the first study showing that the wound-induced VOCs function as a blend to control the biosynthesis of second metabolites at specific concentrations.

Austin (AT) and its derivatives (dehydroaustin (DAT) and acetoxydehydroaustin (ADAT)) produced by Penicillium brasilianum MG-11 exhibit toxicity to insects, yet their targets are unknown. Here, we used whole-cell patch-clamp electrophysiology to investigate the action of AT family compounds on cockroach acetylcholine (ACh), gamma-aminobutyric acid (GABA) and L-glutamate receptors expressed in the American cockroach (Periplaneta americana) neuron. U-tube application of AT or its derivatives did not induce any current amplitudes, suggesting that they did not act as agonist of these three receptors. In the second step of experiments, they were bath-applied for 1 min before co-application with the corresponding ligand. We found that AT and its derivatives had no effect on GABA and L-glutamate-induced currents, whereas they significantly reduced ACh- and epibatidine-induced currents, showing that these compounds acted as selective antagonists of nicotinic acetylcholine receptors (nAChRs) expressed in the cockroach neuron. Of the compounds, DAT showed the highest blocking potency for nAChRs, differentially attenuating the peak and slowly desensitizing current amplitude of ACh-induced responses with pIC(50) (= -log IC(50) (M)) values of 6.11 and 5.91, respectively. DAT reduced the maximum normalized response to ACh without a significant shift in EC(50) , suggesting that the blocking action is not competitive with ACh. (C) 2010 Elsevier Inc. All rights reserved.

Natural pyrethrins biosynthesized by Chrysanthemum cinerariaefolium exhibit insecticidal activity, thereby acting as a defense system against herbivores. Here, we study the effects of mechanical wounding and wound-induced volatile organic compounds (VOCs) on pyrethrin biosynthesis in the seedlings of C. cinerariaefolium. Mechanical wounding resulted in enhanced content of pyrethrins I and II in the seedlings. Wrapping the intact leaves of the wounded seedlings prevented the increase in pyrethrin I but not in pyrethrin II, suggesting that the wound-induced VOCs are involved in the regulation of pyrethrin I biosynthesis, whereas nonvolatile systemic signals play a more important role than the wound-induced VOCs in the regulation of pyrethrin II biosynthesis.

The human alpha 7 nicotinic acetylcholine receptor (nAChR) subunit and its Caenorhabditis elegans homolog, ACR-16, can generate functional recombinant homomeric receptors when expressed in Xenopus laevis oocytes. Both nAChRs express robustly in the presence of the co-injected chaperone, RIC-3, and show striking differences in the actions of a type I positive allosteric modulator (PAM), ivermectin (IVM). Type I PAMs are characterised by an increase in amplitude only of the response to acetylcholine (ACh), whereas type 11 PAMs exhibit, in addition, changes in time-course/desensitization of the ACh response. The type I PAMs, ivermectin, 5-hydroxyindole (5-HI), NS-1738 and genistein and the type 11 PAM, PNU-120596, are all active on human alpha 7 but are without PAM activity on ACR-16, where they attenuate the amplitude of the ACh response. We used the published structure of avermectin B1a to generate a model of IVM, which was then docked into the candidate transmembrane allosteric binding site on alpha 7 and ACR-16 in an attempt to gain insights into the observed differences in IVM actions. The new pharmacological findings and computational approaches being developed may inform the design of novel PAM drugs targeting major neurological disorders. (C) 2009 Elsevier Inc. All rights reserved.

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids.In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO 2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-pi bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-alpha nAChR subunits.

The molecular diversity of many gene products functioning in the nervous system is enhanced by alternative splicing and adenosine-to-inosine editing of pre-mRNA. Using RDL, a Drosophila melanogaster GABA-gated ion channel, we examined the functional impact of RNA editing at several sites along with alternative splicing of more than one exon. We show that alternative splicing and RNA editing have a combined influence on the potency of the neurotransmitter GABA, and the editing isoforms detected in vivo span the entire functional range of potencies seen for all possible edit variants expressed in Xenopus laevis oocytes. The extent of RNA editing is developmentally regulated and can also be linked to the choice of alternative exons. These results provide insights into how the rich diversity of signaling necessary for complex brain function can be achieved by relatively few genes.

Neonicotinoid insecticides are widely used for crop protection based on their selective actions on insect nicotinic acetylcholine receptors (insect nAChRs). Loops C and D in insect nAChRs have been shown to possess structural features favorable for neonicotinoid-nACh R interactions. However, it remains to be resolved whether such features serve either co-operatively, or independently, to enhance neonicotinoid sensitivity of nAChRs. We therefore examined using voltage-clamp electrophysiology the effects on the response to imidacloprid of combinatorial substitutions of residues in loops C and D of the chicken alpha 4 beta 2 nAChR by those present in insect nAChRs. The E219P mutation in loop C of the alpha 4 subunit resulted in enhanced responses to imidacloprid of alpha 4 beta 2, whereas E219S and E219T mutations barely influenced its actions. On the other hand, mutations in loop D (T77R; E79V and T77N; E79R) alone shifted the imidacloprid concentration-response curve to the left (lower concentrations). Interestingly, all three mutations did, however, further enhance the agonist efficacy of imidacloprid when combined with the mutations in loop D. Such synergistic effects of the two loops on the interactions with imidaclprid were observed irrespective of subunit stoichiometry. Computational modeling of the ligand binding domain of the wild-type and mutant alpha 4 beta 2 nAChRs using the crystal structure of the acetylcholine binding protein from Lymnaea stagnalis also indicated that interactions with loop F of loops C and D may contribute to determining the response to imidacloprid. (C) 2008 Elsevier Ltd. All rights reserved.

It has long been known that γ-aminobutyric acid (GABA) participates in a bypass of the tricarboxylic acid (TCA) cycle in plants and bacteria. In a single1950 issue of the Journal of Biological Chemistry, Awapara et al., Roberts and Frankel, and Udenfriend independently reported the presence of a large quantity - approximately 1 mg/g tissue - of GABA in vertebrate brains later similarly high contents were found in the inhibitory neurons of the Atlantic lobster (Kravitz et al. 1963 Otsuka et al. 1967). © Springer Science+Business Media B.V. 2009.

The mode of action and the mechanisms for the selectivity of neurotoxic insecticides were investigated using computational chemistry, molecular biology, homology modeling of the target site and X-ray crystallography. Whole-cell, patch-clamp electrophysiology has been used to show that non-competitive antagonists (NCAs), such as 4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and gamma-hexachlorocyclohexane (gamma-HCH), are more effective blockers of native cockroach (Periplaneta americana) and recombinant house fly (Musca domestica) gamma-aminobutyric acid receptors (GABARs) than glutamate-gated chloride channels (GluCls). Site-directed mutagenesis studies have been used to show that that Ser278 in the second transmembrane region is important for determining the less potent actions of these NCAs on GluCls. The structure-activity relationship and the mechanism of the selectivity of neonicotinoids targeting nicotinic acetylcholine receptors (nAChRs) have also been investigated. Electrophysiological studies show that loops C and D of insect nAChRs are critical in determining the selectivity of neonicotinoids, results consistent with the crystal structures of Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP) in complex with imidacloprid. Neonicotinoids show diverse actions on nAChRs as measured using voltage-clamp electrophysiology. Single channel recording and X-ray crystallography are helping to elucidate the mechanism of super agonist actions of clothianidin and an analog. (C) Pesticide Science Society of Japan

BACKGROUND: Botanical insecticides do not play a major role as crop protectants, but they are beneficial in some applications. The authors investigated the actions of naturally occurring alkaloids on insect nicotinic acetylcholine (ACh) receptors (nAChRs) by evaluating their abilities to inhibit specific binding of [H-3]imidacloprid (IMI) to nerve-cord membranes from Periplaneta americana L. Two alkaloids were also tested for their actions on naAChRs expressed by cockroach neurons using patch-clamp electrophysiology. RESULTS: Four natural quinolizidine alkaloids (matrine, sophocarpine, cytisine and aloperine) exhibited more than 50% inhibition of [H-3]IMI binding at 10 pm, although other compounds were found to have no or low inhibitory activity. The rank order of potency based on concentration-inhibition curves was cytisine > sophocarpine > aloperine > matrine. Patch-clamp analysis indicated that sophocarpine and aloperine were not agonists of nAChRs expressed in P. americana neurons, yet, at 10 gm, aloperine, but not sophocarpine, suppressed ACh-induced inward currents significantly. CONCLUSION: Three of the four natural alkaloids tested possess structural moieties that are necessary for interaction with P. americana nAChRs. Aloperine, which possesses a unique structure and showed a distinctive dose-response curve, was found to act as an antagonist. Appropriate modifications of these alkaloids might result in novel insecticidal nAChR ligands. (C) 2008 Society of Chemical Industry

Neonicotinoid insecticides, which act on nicotinic acetylcholine receptors (nAChRs) in a variety of ways, have extremely low mammalian toxicity, yet the molecular basis of such actions is poorly understood. To elucidate the molecular basis for nAChR-neonicotinoid interactions, a surrogate protein, acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) was crystallized in complex with neonicotinoid insecticides imidacloprid (IMI) or clothianidin (CTD). The crystal structures suggested that the guanidine moiety of IMI and CTD stacks with Tyr185, while the nitro group of IMI but not of CTD makes a hydrogen bond with Gln55. IMI showed higher binding affinity for Ls-AChBP than that of CTD, consistent with weaker CH-π interactions in the Ls-AChBP-CTD complex than in the Ls-AChBP-IMI complex and the lack of the nitro group-Gln55 hydrogen bond in CTD. Yet, the NH at position 1 of CTD makes a hydrogen bond with the backbone carbonyl of Trp143, offering an explanation for the diverse actions of neonicotinoids on nAChRs. © 2008 The Author(s).

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mediating fast cholinergic synaptic transmission in the brain and at neuromuscular junctions. We used the structure of the acetylcholine binding protein from Lymnaea stagnalis to model the chicken alpha 7 agonist-binding domain. The initial models and a preliminary docking study suggested that position Leu118 may play an important role in determining agonist actions on alpha 7. A prediction from these in silico studies, that L118E and L118D would retain binding to acetylcholine but L118K and L118R would not, was confirmed in electrophysiological studies on functional recombinant mutant receptors expressed in Xenopus laevis oocytes. The functional studies also demonstrated that residues at position 118 have a dramatic effect on the actions of imidacloprid (a partial agonist of wild-type alpha 7 receptors) and its des-nitro derivative. Molecular dynamics simulations confirmed that Leu118 can strongly influence agonist binding and that the model was robust in terms of its prediction for acetylcholine binding. Together, the results indicate a role for Leu118 in influencing agonist actions on alpha 7 nAChRs.

The chaperonin GroEL assists protein folding in the presence of ATP and magnesium through substrate protein capsulation in combination with the cofactor GroES. Recent studies have revealed the details of folding cycles of GroEL from Escherichia coli, yet little is known about the GroEL-assisted protein folding mechanisms in other bacterial species. Using three model enzyme assays, we have found that GroEL1 from Chlamydophila pneumoniae, an obligate human pathogen, has a broader selectivity for nucleotides in the refolding reaction. To elucidate structural factors involved in such nucleotide selectivity, GroEL chimeras were constructed by exchanging apical, intermediate, and equatorial domains between E. coli GroEL and C. pneumoniae GroEL1. In vitro folding assays using chimeras revealed that the intermediate domain is the major contributor to the nucleotide selectivity of C. pneumoniae GroEL1. Additional site-directed mutation experiments led to the identification of Gln(400) and Ile(404) in the intermediate domain of C. pneumoniae GroEL1 as residues that play a key role in defining the nucleotide selectivity of the protein refolding reaction.

We synthesized a novel fluorescent analogue of acetylcholine, Cy3-3-acyleholine. The molecular weight of the products agreed with structural predictions. Discrete intensity changes of fluorescent spots due to a single ligand binding/unbinding to nAChR were visualized by TIRF microscopy. The agonist effect of the Cy3-3-acylcholine on nicotinic acetylcholine receptor (nAChR) was confirmed electrophysiologically. This newly synthesized fluorescent analogue will enable us to conduct more elaborate studies on single channel interaction processes between nAChR and ligands. (C) 2007 Elsevier Ltd. All rights reserved.

Chaperonin GroELassists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL- GroES cis-ternary complexes, but prevent the release of METF from GroEL.

Neonicotinoid insecticides target nicotinic acetylcholine receptors (nAChRs), which, in both vertebrates and invertebrates, mediate fast-acting synaptic neurotransmission in the nervous system. Recently, Kagabu et al. synthesized bis-neonicotinoids. The neural activities of bis-neonicotinoids have been evaluated on the central nerve cord of American cockroaches. However, the action of bis-neonicotinoids on nAChRs expressed by dissociated insect neurons has not yet been studied. Thus, the actions of several alkylene-tethered bis-neonicotinoids on the terminal abdominal ganglion neurons of the American cockroach, Periplaneta americana, were investigated using whole-cell patch-clamp electrophysiology. All of the ligands tested did not induce membrane currents, but reduced the responses to ACh when bath applied prior to co-application with ACh. Of the compounds tested, HK- 13, which possesses two imidacloprid units linked with a hexamethylene bridge, had the highest antagonist potency. The antagonist action was reduced, not only by elongating, but also by shortening the linker. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Various bacterial species were isolated from the crop (digestive organ) of the antlion species Myrmeleon bore and tested for their insecticidal activity against caterpillars by injection. Sixty-eight isolates from the antlion crop were grouped into twenty-four species based on homologies of 16S rRNA gene sequences and biochemical properties. Isolated Bacillus cereus, Bacillus sphaericus, Morganella morganii, Serratia marcescens and a Klebsiella species killed 80% or more cutworms when injected at a dose of 5 x 105 cells per insect. In addition, cutworms killed by these isolates resembled observations made of caterpillars attacked by antlions. A culture-independent analysis showed that the isolated bacterial species are likely to be frequently present in the antlion crop. These results suggest that insecticidal microorganisms associate with antlions, and may promote the death of prey. (c) 2007 Elsevier Inc. All rights reserved.

Prod rugs of imidacloprid and the thiazolylmethyl analog masked with oxodioxolylniethyl group on the N3 site were prepared. The prodrugs decomposed in a buffer solution of pH 8.3 and in a physiological salt solution with half-lives of 10-15 h, releasing the parent insecticides. Being consistent with this, an inward current was evoked in dissociated cockroach neurons treated with the masked compound solutions, which were maintained for 24 h after preparation, as measured using patch-clamp electrophysiology, whereas no response was observed in neurons when the solutions were challenged immediately after preparation. The insecticidal test on the American cockroach showed that the minimum lethal dose for each compound at 24 h after injection was 6.4 x 10(-8) mol, which was similar to that for imidacloprid and the thiazolyl derivative. This result strongly suggested a regeneration of the active ingredients in vivo. (c) 2007 Elsevier Ltd. All rights reserved.

An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's pore-forming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.

The low mammalian toxicity of neonicotinoid insecticides has been shown to be attributable, at least in part, to their selective actions on insect nicotinic acetylcholine receptors (nAChRs). There are multiple nAChRs in insects and a wealth of neonicotinoid chemicals. Studies to date have discribed a wide range of effects on nAChRs, notably partial agonist, super agonist and antagonist actions. Both the diversity of the neonicotinoid actions and their selectivity for insect over vertebrate nAChRs are the result of physicochemical and steric interactions at their molecular targets (nAChRs). In such interactions, the formation and breakage of hydrogen bond (HB) networks plays a key role. Therefore the loss or gain of even a single HB resulting from either structural changes in neonicotinoids, or the amino acid sequence of a particular nAChR subunit, could result in a drastic modification of neonicotinoid actions. In addition to the amino acid residues, the backbone carbonyl of nAChRs may also be involved in the formation of HB networks with neonicotinoids. © 2007 Springer-Verlag.

The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha 4 beta 2 nAChR and Drosophila melanogaster D alpha 2/chicken beta 2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta 2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha 4 beta 2 nAChR, T77R; E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K; E79R and T77N; E79R double mutations in the D alpha 2 beta 2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha 4 beta 2 and D alpha 2 beta 2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC50 value for ACh of 19 mu M. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 mu M ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the -1′ position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken α7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing α7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the α7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans. © Springer-Verlag 2006.

Many of the insecticides in current use act on molecular targets in the insect nervous system. Recently, our understanding of these targets has improved as a result of the complete sequencing of an insect genome, i.e., Drosophila melanogaster. Here we examine the recent work, drawing on genetics, genomics and physiology, which has provided evidence that specific receptors and ion channels are targeted by distinct chemical classes of insect control agents. The examples discussed include, sodium channels (pyrethroids, p,p′- dichlorodiphenyl-trichloroethane (DDT), dihydropyrazoles and oxadiazines) nicotinic acetylcholine receptors (cartap, spinosad, imidacloprid and related nitromethylenes/nitroguanidines) γ-aminobutyric acid (GABA) receptors (cyclodienes, γ-BHC and fipronil) and L-glutamate receptors (avermectins). Finally, we have examined the molecular basis of resistance to these molecules, which in some cases involves mutations in the molecular target, and we also consider the future impact of molecular genetic technologies in our understanding of the actions of neuroactive insecticides. © Springer-Verlag 2005.

4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl- channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC50 values under steady state condition. The IC50 values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH. © Springer-Verlag 2005.

A chimera based on the chicken alpha 4 nicotinic acetylcholine receptor (nAChR) subunit containing an insert from loop B to the N-terminus of the Drosophila melanogaster D alpha 2 (=SAD) subunit was constructed and co-expressed with the chicken beta 2 nAChR subunit in Xenopus laevis oocytes. The actions of the neonicotinoid insecticide imidacloprid were examined. Replacement of the region loop B to the N-terminus of the alpha 4 subunit by the corTesponding region of the D alpha 2 subunit had little effect on the concentration-response curve for imidacloprid. However, replacement of Glu219 by proline in the YXCC motif in loop C of the chimeric alpha 4 subunit resulted in a marked displacement to the left of the concentration-response curve for imidacloprid not seen when an equivalent mutation was made in the alpha 4 beta 2 nAChR. The results suggest that the region loop B to the N-terminus in the D alpha 2 subunit contributes to the high imidacloprid sensitivity of the hybrid D alpha 2 beta 2 nAChR. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Neonicotinoid insecticides, which act selectively on insect nicotinic acetylcholine receptors (nAChRs), are used worldwide for insect pest management. Studies that span chemistry, biochemistry, molecular biology, and electrophysiology have contributed to our current understanding of the important physicochemical and structural properties essential for neonicotinoid actions as well as key receptor residues contributing to the high affinity of neonicotinoids for insect nAChRs. Research to date suggests that electrostatic interactions and possibly hydrogen bond formation between neonicotinoids and nAChRs contribute to the selectivity of these chemicals. A rich diversity of neonicotinoid-nAChR interactions has been demonstrated using voltage-clamp electrophysiology. Computational modeling of nAChR-imidacloprid interaction has assisted in the interpretation of these results.

The biosynthetic pathway to natural pyrethrins in Chrysanthemum cinerariaefolium seedlings was studied using [1-C-13]D-glucose as a precursor, with pyrethrin I isolated using HPLC from a leaf extract. The C-13 NMR spectrum of pyrethrin I from the precursor-administered seedlings indicated that the acid moiety was biosynthesized from D-glucose via 2-C-methyl-D-erythritol 4-phosphate, whereas the alcohol moiety was possibly biosynthesized from linolenic acid. (c) 2005 Elsevier Ltd. All rights reserved.

Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs), but little is known about the mechanism of selectivity. To elucidate the mechanism, structural features of neonicotinoids and insect nAChRs contributing to this selectivity have been examined. Using molecular-oribital calculations, electrostatic interactions and hydrogen-bond formation of neonicotinoids with insect nAChRs were postulated to contribute to the selectivity of neonicotinoid-nAChR interactions. Also, the use of voltage-clamp electrophysiology combined with molecular biology showed that replacement of the vertebrate alpha 4 subunit in the alpha 4 beta 2 nAChR by Drosophila alpha subunits and mutation to basic residues of an amino acid in loop D of the alpha 7 nAChR enhanced neonicotinoid sensitivity of the nAChRs. These findings suggest important roles for alpha and non-alpha subunits in the selective actions of neonicotinoids on insect nAChRs.

Atherosclerosis is a vascular disorder involving inflammation, a narrowed vascular lumen in the entire tunica intima, and reduced elasticity of the arterial wall. It has been found that Hsp60 from Chlamydia pneumoniae, an obligate bacterial pathogen associated with atheroma lesions, mimics human Hsp60, thereby causing attacks by immune cells on stressed endothelial cells expressing endogenous Hsp60 on their surface. Furthermore, Hsp60 from C. pneumoniae has been shown to promote the growth of vascular smooth muscle cells (VSMCs). To explore probes that can be used for studying signal transduction elicited by the chlamydial Hsp60, we have tested several natural products for their inhibitory actions on the Hsp60-induced proliferation of rat arterial smooth muscle cells. Sesamol, vanillyl alcohol, and trans-ferulic acid exhibited moderate inhibitory actions on the Hsp60-induced cell proliferation; zerumbone, humulene, and caryophylene effectively inhibited it at low concentrations with IC(50) values of 529, 122, and 110 nM, respectively. The results indicated that the 11-membered alicyclic ring is favorable for interactions with receptors involved in the Hsp60-induced VSMC proliferation.

A calcium-alginate gel diet was developed for Spodoptera litura larvae, and its reliability as a carrier for incorporating antifeedants as well as insecticides was investigated. The alginate gel diet was prepared with a simple protocol, which does not involve any heating process. When tested using this diet, acephate, a Bacillus thuringiensis endotoxin formulation and rotenone reproducibly showed insecticidal activity against the larvae, while neem oil and scabequinone deterred the larval feeding effectively. However, not only the insecticidal activity of acephate but also the antifeedant activity of neem oil was reduced by replacing the alginate component by agar in the diet, suggesting the usefulness of the alginate gel diet as an assay tool for testing a broad range of samples against the larvae.

To elucidate the mechanism of selective action of imidacloprid on insect nicotinic acetylcholine receptors (nAChRs), we examined the roles of loop C and the loop B-C interval region in receptor interactions with imidacloprid. The P242E mutation in loop C of the Drosophila SAD subunit (the second alpha-like Drosophila nicotinic acetylcholine receptor subunit, also called Dalpha2 subunit) reduced imidacloprid sensitivity of the SAD-chicken beta2 hybrid nAChR, whereas the E219P mutation of the alpha4 subunit increased the imidacloprid sensitivity of the alpha4beta2 nAChR. Deletion of the loop B-C interval region from the SAD subunit enhanced the effect of the P242E mutation on the SADbeta2 hybrid nAChR, suggesting important roles of the regions investigated in the nAChR-imidacloprid interactions. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SADbeta2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.

Bacillus cereus isolated from the larvae of Myrmeleon bore was found to secrete proteins that paralyze and kill German cockroaches, Blattela germanica, when injected. One of these active proteins was purified from the culture broth of B. cereus using anion-exchange and gel-filtration chromatography. The purified toxin, with a molecular mass of 34 kDa, was identified as sphingomyelinase C (EC 3.1.4.12) on the basis of its N-terminal and internal amino-acid sequences. A recombinant sphingomyelinase C expressed in Escherichia coli was as potent as the native protein in killing the cockroaches. Site-directed mutagenesis (His151Ala) that inactivated the sphingomyelinase activity also abolished the insecticidal activity, suggesting that the rapid insect toxicity of sphingomyelinase C results from its phospholipid-degrading activity.

The nitro group of a neonicotinoid, imidacloprid, plays a key role in its selective actions on insect nicotinic acetylcholine receptors (nicotinic AChRs) and is postulated to bind close to residues Q79 in loop D and G189 in loop F of the chicken alpha7 nicotinic AChR. To evaluate the relative contributions of these residues to interactions with imidacloprid, Q79 and G 189 were replaced in tandem by first basic then acidic residues. Changes in the currents evoked by imidacloprid and acetylcholine (ACh) on the alpha7 wild type and mutant receptors expressed in Xenopus laevis oocytes were investigated using two-electrode voltage clamp electrophysiology. An increase in the efficacy of imidacloprid for the alpha7 receptor resulting from the Q79K and Q79R mutations was suppressed by a G189E mutation in loop F. However, the increases in efficacy resulting from such Q79 mutations were scarcely influenced by a G189D substitution. Three-dimensional modeling of the alpha7 nicotinic AChR, based on the acetylcholine-binding protein (AChBP) of Lymnaea stagnalis, suggests that the reduced efficacy of imidacloprid following the G189E mutation is likely to result from carboxylate interference with the electronic interactions between the nitro group of imidacloprid and the basic residues in loop D. (C) 2003 Elsevier B.V. All rights reserved.

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha 7, chicken alpha 4 beta 2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta 2 and ALS/beta 2. No agonist action of NTX (0.1-100 mu M) was observed on chicken alpha 7, chicken alpha 4 beta 2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha 7) or heteromeric (alpha 4 beta 2) nicotinic AChRs. Block by NTX of the chicken a7, chicken alpha 4 beta 2 and the SAD/beta 2 and ALS/beta 2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.

The electrophysiological actions of various neonicotinoids, including substituted benzyl derivatives, against recombinant Drosophila SAD/chicken beta2 hybrid nicotinic acetylcholine receptor (nAChR) were measured to analyze the relationships between the in vivo (insecticidal) and in vitro (binding and agonist) activities. Most of the neonicotinoids tested were capable of inducing inward currents by activating the hybrid nAChRs expressed in Xenopus laevis oocytes, whereas some compounds had no agonist activity and only blocked the acetylcholine-induced currents. Variations in the agonist activity were well correlated with those in the binding potency evaluated using [H-3]imidacloprid as well as insecticidal activities. (C) 2003 Society of Chemical Industry.

The 2-nitroimino-imidazolidine and related moieties are structural features of neonicotinoid insecticides acting on nicotinic acetylcholine receptors (nicotinic AChRs). To evaluate these moieties in neonicotinoid interactions with nicotinic AChR alpha subunits, the actions of imidacloprid and related compounds on the chicken alpha7, alpha4beta2 and Drosophila melanogaster-chicken hybrid (SADbeta2 and ALSbeta2) receptors expressed in Xenopus laevis oocytes were studied by voltage-clamp electrophysiology. Imidacloprid and nitenpyram were partial agonists and a nitromethylene analog of imidacloprid (CH-IMI) was a full agonist of the beta receptor, whereas their agonist actions on the alpha4beta2 receptor were very weak, contrasting with full agonist actions of DN-IMI, a desnitro derivative of imidacloprid. The neonicotinoids and DN-IMI were either full or partial agonists of the SADbeta2 receptors. Nitenpyram and DN-IMI were partial agonists of the ALSbeta2 receptor, whereas imidacloprid and CH-IMI scarcely activated the ALSbeta2 receptor. Imidacloprid and CH-IMI in fact suppressed ACh-induced responses of the ALSbeta2 receptor, whereas imidacloprid potentiated and CH-IMI suppressed ACh-induced responses of the alpha4beta2 receptor. These results suggest that interactions with alpha subunits of the 2-nitroimino-imidazolidine moiety of imidacloprid play a role in determining not only agonist and antagonist actions on all four receptors, but also the potentiation of ACh-induced responses of the alpha4beta2 receptor. (C) 2003 Elsevier Science Ltd. All rights reserved.

Neonicotinoid insecticides are agonists of insect nicotinic acetylcholine receptors (AChRs) and show selective toxicity for insects over vertebrates. To elucidate the molecular basis of the selectivity, amino acid residues influencing neonicotinoid sensitivity were investigated by site‐directed mutagenesis of the chicken α7 nicotinic AChR subunit, based on the crystal structure of an ACh binding protein (AChBP). In the ligand binding site of AChBP, Q55 in loop D is close to Y164 in loop F that corresponds to G189 of the α7 nicotinic receptor. Since Q55 of AChBP is preserved as Q79 in the α7 nicotinic receptor and the G189D and G189E mutations have been found to reduce the neonicotinoid sensitivity, we investigated effects of Q79E, Q79K and Q79R mutations on the neonicotinoid sensitivity of the α7 receptor expressed in Xenopus laevis oocytes to evaluate contributions of the glutamine residue to nicotinic AChR–neonicotinoid interactions. The Q79E mutation markedly reduced neonicotinoid sensitivity of the α7 nicotinic AChR whereas the Q79K and Q79R mutations increased sensitivity, suggesting electronic interactions of the neonicotinoids with the added residues. By contrast, the Q79E mutation scarcely influenced responses of the α7 nicotinic receptor to ACh, (−)‐nicotine and desnitro–imidacloprid (DN–IMI), an imidacloprid derivative lacking the nitro group, whereas the Q79K and Q79R mutations reduced the sensitivity to these ligands. The results indicate that the glutamine residue of the α7 nicotinic receptor is likely to be located close to the nitro group of the insecticides in the nicotinic receptor–insecticide complex. British Journal of Pharmacology (2002) 137, 162–169. doi:10.1038/sj.bjp.0704848

The nitroguanidine insecticide imidacloprid along with a second generation of related compounds including nitenpyram, all nicotinic acetylcholine (ACh) receptor ligands, are used increasingly in many countries. Site‐directed mutagenesis and heterologous expression in Xenopus laevis oocytes have been deployed to investigate mutants (G189D and G189E) of the chicken α7 homomer‐forming nicotinic receptor subunit which are predicted to enhance the negative charge at the negative subsite (loop D) of the ACh binding site. Xenopus oocytes expressing wild‐type α7 nicotinic receptors respond to imidacloprid with rapid inward currents. Imidacloprid and nitenpyram are partial agonists, whereas ACh, (−)‐nicotine and (+)‐epibatidine are full agonists. Compared to wild‐type α7, the mutant G189D and G189E receptors are much less sensitive to the insecticides, whereas their sensitivity to (−)‐nicotine, ACh and (+)‐epibatidine is only slightly reduced. In contrast, G189N and G189Q mutants are sensitive not only to ACh, (−)‐nicotine and (+)‐epibatidine, but also to the two insecticides. Thus reduction of the insecticide‐sensitivity by the mutations G189D and G189E are attributed to an increase in negativity of loop D. Desnitro‐imidacloprid (DN‐IMI), an imidacloprid derivative lacking the nitro group is a potent agonist on the G189D and G189E mutants suggesting an important role of loop D in nicotinic receptor interactions with the nitro group of nitroguanidine insecticides. British Journal of Pharmacology (2000) 130, 981–986; doi:10.1038/sj.bjp.0703374

A novel tricyclic dinitrile, KN244, blocked the wild‐type (dieldrin‐sensitive) homo‐oligomeric γ‐aminobutyric acid (GABA)‐gated chloride channel of Drosophila melanogaster expressed in Xenopus oocytes. Sensitivity to the block by KN244 of the response to 30 μM GABA (IC₅₀=41.6 nM, wild‐type RDL_ac) was reduced abut 100 fold (IC₅₀=4.5 μM) in the dieldrin‐resistant (RDL_ac^A302S) form of RDL. British Journal of Pharmacology (1999) 127, 1305–1307; doi:10.1038/sj.bjp.0702663

3-Indolepropionic acid (IPA)-related compounds having a benzo[b]thiophene or an indazole ring and derivatives having various substituents in the propionic acid moieties were tested for their antibacterial activity against Ralstonia solanacearum. Substitution of the indole ring for other aromatic rings resulted in lowered activity, whereas addition of a methyl or a trifluoromethyl group to the propionic acid moiety had little effect. Of the derivatives, 3-(3-indolyl)butanoic acid (3-IBA) was as active as IPA, exhibiting a 10-fold higher activity with the S configuration than with the R configuration. In contrast with the strong phytotoxicity of IPA, 3-IBA was able to suppress bacterial wilt without affecting the growth of tomato plants.

Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co‐expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)‐epibatidine, (−)‐nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two‐electrode, voltage‐clamp electrophysiology. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)‐epibatidine, (−)‐nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)‐nicotine, whereas (+)‐epibatidine and acetylcholine were full agonists. The EC₅₀ of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC₅₀ for (+)‐epibatidine, (−)‐nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh‐mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit. British Journal of Pharmacology (1998) 123, 518–524; doi:10.1038/sj.bjp.0701618

We show that three of the eleven genes of the nematodeCaenorhabditis elegansthat mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits.unc-38encodes an α subunit whilelev-1andunc-29encode non-α subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression inXenopusoocytes of combinations of these subunits that include theunc-38α subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, andd-tubocurarine but not α-bungarotoxin. The mutant phenotypes reveal thatunc-38andunc-29subunits are necessary for nAChR function, whereas thelev-1subunit is not. An UNC-29–GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations oflev-1result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants inC. elegansprovides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studiedin vivo.

A photoreactive and fluorescent ester, named “KUP-1,” was synthesized as a possible probe for studies at the primary site of action of pyrethroids. Nerve effects of the compound were fundamentally the same as those induced by conventional pyrethroids. KUP-1 induced the depolarizing afterpotential in the falling phase of the action potential and depolarized the resting membranes in crayfish and cockroach giant axons. The compound slowed the decay of the sodium currents measured in the crayfish giant axon under the voltage-clamp conditions. The maximum wavelength of the emission spectrum was apart enough to distinguish the compound from proteins, indicating that the compound could be used as a tool for photo-affinity labeling. © 1995, Pesticide Science Society of Japan. All rights reserved.

1. W-7, an anticalmodulin agent, prolonged the falling phase of the action potential in crayfish (Procambarus clarkii) giant axons, as did meta-methylbenzyl pyrethrate, a pyrethroid insecticide, and the methoxychlor of a DDT analog. 2. The anticalmodulin agent suppressed both the inward and outward membrane currents in the axon, measured under voltage-clamp conditions. 3. The pyrethroid and methoxychlor insecticides induced a large inward tail-current upon step repolarization, but the anticalmodulin agent did not induce a current of a detectable size. © 1993.

熱失活や昆虫試料による供試化合物の変性を低減する為に、 アルギン酸カルシウムゲルを用いた昆虫飼料を成型・給餌する生物検定法を提案し、 本法を用いた数種の活性既知化合物の評価を行った。

本研究の目的は、昆虫の性別、生育段階および神経組織の違いによるニコチン性アセチルコリン受容体（nAChR）のサブユニット構成とそれを制御する補助タンパク質因子をつぶさに明らかにし、それぞれのnAChRに対する殺虫剤ネオニコチノイドおよび関連殺虫剤、さらにはnAChRを標的とする微生物産物等の作用機構を明らかにすることである。これまでに、ショウジョウバエ由来のサブユニットが形成するnAChRをアフリカツメガエル卵母細胞に網羅的かつ機能的に発現させ、電気生理学的手法の一種である膜電位固定法を用いて、それぞれのnAChRに対するネオニコチノイドの活性を評価した。また、当該活性の変動に対するサブユニット構成比や機能的発現を補助するタンパク質の寄与の程度を明らかにした。また、セイヨウミツバチの神経組織の違いに応じたnAChRのサブユニット構成や補助タンパク質因子の変化について明らかにした。さらに、その他の昆虫種についてnAChRの機能的発現技術について検討するとともに、昆虫と同じく機能的発現に補助因子を必要とする無脊椎生物のnAChRに選択的に作用する微生物二次代謝物質や、nAChRに類する構造をもつ昆虫のリガンド作動性イオンチャネルにおける微生物二次代謝物質の活性発現機構についても明らかにした。 一方、本研究を進めるためのケミカルツールとして昆虫脱皮ホルモンであるエクジソンの生合成を選択的に調節する糸状菌二次代謝物質の構造活性相関を解明した。

LaIT1はヤエヤマサソリ毒液から単離された昆虫選択的ペプチド毒素である。本研究においては、光親和性標識されたLaIT1類縁体を合成し、これを用いてカイコ幼虫の中枢神経においてLaIT1と特異的に結合するタンパク質の探索を行った。その結果、候補として得られたタンパク質はこれまでにサソリ毒の作用標的として報告されていないことから、LaIT1は新しい作用機構を持つ殺虫性ペプチドであることが示唆された。

地球規模での安定した食料生産は重要な課題である。農薬は、農作物を病害虫から保護するための必須な農業資材であり、食料生産に大きな貢献している。本研究では、昆虫や線虫の体内に存在するリガンド作動性陰イオンチャネルの機能と性質を調べ、安全な有害生物防除剤の作用点としてのこのチャネルの可能性を検証する研究を行った。その結果、新規作用機構解明、薬剤創製に導く新規化合物の発見、新規標的イオンチャネルの同定・解析などを行うことができた。

相関係数値と幾何学的要素（外側と内側の線の繋がり方）で相関ネットワーク解析を行う金平糖アルゴリズムを搭載したConfeitoGUIplus ソフトを開発した。本ソフトは多変量解析の1種である。シロイヌナズナの公開DNAマイクロアレイを活用し、フラボノ-ル生合成のモジュールを得ることを示した。 除虫菊(Tanacetum cinerariifolium)が生合成する天然殺虫剤成分であるピレスリン生合成遺伝子情報を網羅的獲得する目的でRNA-Seq解析を行った。得られた数種のピレスリン生合成遺伝子発現に対して、傷害誘導的に生じる揮発性有機化合物(VOC) のブレンド作用効果を調査した。

エステル構造をもつ殺虫性物質ピレスリンは、除虫菊の中で酸部とアルコール部とがGDSL リパーゼTcGLIPのはたらきにより連結されることにより生合成される。本研究では、本酵素の制御機構を解明するため、まずその触媒機構に必須のアミノ酸を大腸菌で発現させた組換え酵素を用いて同定し、結晶化に適した塩類溶液を明らかにした。また、低濃度で不可逆的に本酵素を阻害するホスホン酸エステル類を創出し、本酵素の遺伝子発現が傷害誘導的に生じる揮発性物質により促進されることを明らかにした。さらに、シロイヌナズナで本酵素遺伝子のホモログ遺伝子を破壊すると、防御遺伝子のプロモータ促進活性が低下することを見出した。

ウンカ類の薬剤抵抗性発達程度には種や地域差があり、ベトナム南部のトビイロウンカで抵抗性が極めて高かった。一方、遺伝子多様性からはウンカ類の地域的特徴が明確ではなかった。イミダクロプリド抵抗性はCYP6ER1遺伝子に起因する酵素活性の増加によって起こると考えられた。移動解析から、トビイロウンカの東アジアと東南アジア間の個体群境界を越えた移動、およびベトナム南部では比較的近距離の移動の可能性が示唆された。

イベルメクチン(IVM)やミルベマイシン(MLM)等のマクロライド系化合物による抑制性グルタミン酸受容体(GluCl)の活性化機構解明するため、化合物の結合部位の解明に有用な光反応性試薬を開発した。また、カイコGluClの結晶化に必須の大量発現方法を確立した。さらに捻転胃虫GluClに対するMLM-A_4の活性発現に重要なアミノ酸を同定し、本化合物の結合部位が2か所存在する可能性を見出した。

本研究では(1)植物の香りの生合成経路であるフィトオキシリピン経路の間接防衛に果たす役割の全体像の解明とその応用の研究から、みどりの香りの生態機能に関する多くの新知見を得た。とくに除虫菊の研究から新たな植物防衛の機構が明らかになった。また(2)植物の揮発性物質が生態系の生物間相互作用ネットワークに及ぼす影響の解明とその応用に関する研究では、相互作用・情報ネットワークの概念を確立するとともに、揮発性物質の利活用による害虫防除法を発見した。

本研究者は,脈翅目ウスバカゲロウ科昆虫に属するクロコウスバカゲロウ幼虫には殺虫性蛋白質を産生する細菌類が共生する.本研究では,それらの共生細菌類が産生する殺虫性蛋白質の構造を解明するとともに,蛋白質の諸性質を明らかにすることを目的とした.本研究の開始時点では,殺虫性蛋白質を産生する細菌としてEnterobacter aerogenesが得られていた.しかし殺虫性蛋白質を産生するクロコウスバカゲロウ幼虫の共生細菌はE. aerogenesだけではないと考え,同じような性質を示す共生細菌を探索した結果,Basillus cereusを見出した.本菌は,殺虫活性を示すphospholipase C (PLC)を分泌することで知られている.そこで,クロコウスバカゲロウ幼虫の吐き戻し液にPLCが含まれているのかどうか調べた結果,吐き戻し液から一定の割合でPLCが検出された.PLCとは異なる殺虫性蛋白質をB.cereusの培養液から精製した結果,分子量約70Kの蛋白質Toxin 1および分子量約35Kの蛋白質Toxin 2を単離した.Toxin 1および2は,それぞれzinc methalloprotease inh2Aおよびsphingomyelinase Cと相同性を示した.両蛋白質をコードする遺伝子をB.cereusからクローニングして塩基配列を解読することにより,それらの全一次構造を解明した.一方,クロコウスバカゲロウ幼虫の素嚢から共生細菌類を単離し,ハスモンヨトウ幼虫に対して経口的に投与した.その結果,多くの細菌類がハスモンヨトウ幼虫に対して顕著な感染性を示し,投与開始後数日以内に幼虫を死滅させることを見出した.以上のような研究と併行して,殺虫性蛋白質の試験系の一つとして,ネオニコチノイド系殺虫剤に対して様々な感受性を示すニコチン性アセチルコリン受容体をも構築した.

新規化合物の新たな探索技術法は,害虫を防除する手段として主要に用いられている殺虫剤の開発の場面で強く望まれている.特に環境中に放出する化学物質に対する安全性に対する配慮,新規化合物の効率的な開発とその高活性化,従来の殺虫剤に対して抵抗性を発達させた害虫に対する高活性など多くの条件を備えた物質を持続的に開発していくことが求められている.化合物の作用機構に基づいた視点からの新規化合物の開発技術は,近年可能性として論じられてきたが,実際の検討は特に殺虫剤についてはまだ不十分である.本研究では、神経のイオンチャネルに作用する殺虫剤の作用機構を電気生理学,生化学,分子生物学のそれぞれの手法を用いて検討し,新規化合物の発見が効率的に行われるための基礎研究を行った. アセチルコリンレセプターを作用点とする殺虫剤としてイミダクロプリド、アセタミプリド、GABAレセプターを作用点とする殺虫剤としてフィプロニル、さらに天然物であるアニサチンについて作用機構を調べた.その結果、イミダクロプリドおよびアセタミプリドはアセチルコリンレセプターに対して部分アゴニストとして作用することが明らかとなった.このことは、新規殺虫剤の開発の場面においてアゴニストの探索のツールとして、これらの化合物を用いることで、同種グループの殺虫剤を効率的に開発することが可能になると同時に、これらと同様の作用メカニズムを持つ別の殺虫剤との交差抵抗性の発達を防ぐための重要な知見になると思われる.フィプロニルとアニサチンは相互に作用メカニズム面では共通点があるが、異なる点があることが示された.特にイオンチャネルのステートに対して異なった作用を示すことから、アニサチンを親化合物として、新規化合物の探索を行うことで、新規化合物を開発することが可能であることが示された. 以上の研究より、分子レベルの電気生理学的手法が、殺虫剤の探索技術として用いることが可能であることが示された.今後はさらに膜電位感受性色素などを用いた光学的手法を組み合わせることにより、より実践的な探索技術の開発が可能になるであろうと思われる.

クロコウスバカゲロウ幼虫の唾液には、数種の殺虫性蛋白質が含まれている。本研究では、これらの殺虫性蛋白質をコードする遺伝子をクローニングし、組換え蛋白質を大腸菌で発現させ、その選択毒性を評価しようとした。クロコウスバカゲロウ幼虫の唾液に含まれている殺虫性蛋白質は、幼虫自身がつくるものと考えて研究を進めたが、目的とする遺伝子をクローニングすることはできなかった。そこで、それらの殺虫性蛋白質はクロコウスバカゲロウ幼虫がつくるのではなく、共生微生物がつくるのではないかと推定し、本幼虫の唾液から単離した共生細菌類を特定の条件下で培養した。その結果、共生細菌は予想通り殺虫性蛋白質を産生することが明らかとなった。共生細菌の培養液から単離した一殺虫性成分の部分アミノ酸配列を解明したところ、本成分はシャペロニンとして知られるGroELのホモログであることが示唆された。さらに、共生細菌からクローニングしたgroEL遺伝子を大腸菌で発現させたところ、組換え蛋白質は共生細菌の培養液から単離した蛋白質と同程度の殺虫活性を示したことから、共生細菌が産生する殺虫性成分の一つはGroELホモログであると考えた。本組換え蛋白質は、各種の昆虫類に対して幅広く殺虫活性を示したが、マウスに対しては顕著な毒性を示さなかった。一方大腸菌のGroELは、マウスのみならず昆虫に対しても毒性を示さなかった。クロコウスバカゲロウ幼虫の共生細菌が産生するGroELホモログと大腸菌のGroELのアミノ酸配列は、C末端の数残基を除くと11残基異なっていた。そこで、共生細菌のGroELホモログのアミノ酸残基を大腸菌型に、一方大腸菌のGroELのアミノ酸残基を共生細菌のGroELに見られるアミノ酸残基に変化させ、殺虫活性の変化を調べた。その結果、共生細菌のGroELホモログの昆虫毒性に深く関与する4つのアミノ酸残基を決定することができた。

1、ピレスロイド、メトキシクロル、およびカルモジュリン阻害剤の神経生理活性の比較ピレスロイドやDDTはカルモジュリン依存性の酵素群を阻害することを報じた論文を参考にして、数種のピレスロイド、メトキシクロル(DDT類縁体)、およびW-7を代表とするカルモジュリン阻害剤のアメリカザリガニ巨大軸索膜に対する効果を検討した。膜電位レベルでは、殺虫剤およびW-7は活動電位の下降相に互いに良く似た形状の脱分極性後電位を誘起したが、W-7以外のカルモジュリン阻害剤は脱分極性後電位を誘起しなかった。膜電位固定法によって殺虫剤とW-7の膜電流におよぼす効果を解析したところ、殺虫剤はナトリウムチャネルの不活性化を遅らせるのに対し、W-7はカリウム電流の増大を抑制することがわかった。これらの事実より、ピレスロイドとDDTの作用はカルモジュリンを介して発現するのではないと推定した。2、ピレスロイドとしての活性を有する光親和性標識化合物の合成ピレスロイドのレセプターを究明するため、酸部分を3-methyl-2-(4-azidophenyl)butanoic acidとし、アルコール部分をN-hydroxymethyl-4-methoxyphthalimideとするエステルを合成した。化合物はシュークロースギャップ法で測定されるアメリカザリガニ巨大軸索膜のナトリウム電流の不活性化過程を阻害し、パッチクランプ法によって観察される種々の培養神経細胞のナトリウム電流の開状態時間を引き伸ばすことを確認した。以上の生理活性は、これまでに報告されているピレスロイドの生理活性と同様のものであることから、新たに合成した化合物はピレスロイドと同じレセプターに作用するものと考えた。今後、本化合物の酸部分が持つ光親和性官能基とアルコール部分が発する蛍光を利用して、ピレスロイドのレセプターを同定する予定である。

植物病害防徐新戦略構築へのバイオテクノロジー技術の多面的応用について検討した。1.土壌中病原菌と細菌の拮抗現象を利用した菌類病防徐システムを開発した。キチン分解性細菌の生産するキチナーゼを精製し、分解様式を明らかにするとともに、それら遺伝子をクローニングし、塩基配列を決定した。ビーズに固定した分解菌をキチン・ゼオライト添加士壌に施用すると、フザリウム菌は減少し、トマト萎凋病、イチゴ萎黄病の防徐効果が認められた。2.フザリウム菌、うどんこ病菌用のベクターを開発し、マイクロインジェクション(MI)またはエレクトロポーレーション法によって胞子に導入し、これらの菌の形質転換に成功した。菌類病原性解析の基礎条件を確立した。3.宿主細胞への外来遺伝子の導入と発現解析の条件を明らかにした。MI法を基本技術とし、転写産物はin situハイブリダイゼーションにより、また翻訳産物は細胞化学的呈色反応または免疫化学法によって検出した。感染細胞で特異的に発現する遺伝子を分割スクリーニング法によって特定する技術を開発し、宿主・病原体相互反応を遺伝子発現のレベルで解析する手法を確立した。4.木材腐朽菌によるリグニン分解の分子機構を酵素学的に解析し、その遺伝子のクローニングを試みた。5.トマト青枯病をバクテリオファージを用いて防除するための基礎を確立した。ファージ系統の宿主識別に関わる外被タンパク遺伝子をクローニングするとともに。有効なプロモーターを分離した。6.組織培養系を利用した体細胞変異選抜から、タバコモザイクウイルス並びに青枯病抵抗性トマト、萎黄病並びに炭そ病抵抗性イチゴ系統を作出した。トマトでは後代検定も終え、実用化試験を行っている。7.アグロインフェクションによってキュウリモザイクウイルス外被タンパク遺伝子をメロンに導入し、同ウイルス抵抗性形質転換体を作出した。