Functional impact of subunit composition and compensation on Drosophila melanogaster nicotinic receptors-targets of neonicotinoids.

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, Dß1, and Dß2 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 Dß3 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 Dß3 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.

Investigation Of The Biosynthesis Pathway That Generates cis-Jasmone.

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.

Understanding pyrethrin biosynthesis: toward and beyond natural pesticide overproduction.

Pyrethrins are natural insecticides biosynthesised by Asteraceae plants, such as Tanacetum cinerariifolium and have a long history, dating back to ancient times. Pyrethrins are often used as low-persistence and safe insecticides to control household, horticultural, and agricultural insect pests. Despite its long history of use, pyrethrin biosynthesis remains a mystery, presenting a significant opportunity to improve yields and meet the growing demand for organic agriculture. To achieve this, both genetic modification and non-genetic methods, such as chemical activation and priming, are indispensable. Plants use pyrethrins as a defence against herbivores, but pyrethrin biosynthesis pathways are shared with plant hormones and signal molecules. Hence, the insight that pyrethrins may play broader roles than those traditionally expected is invaluable to advance the basic and applied sciences of pyrethrins.

Divergent Nine-Step Syntheses of Perhydrohistrionicotoxin Analogs and Their Inhibition Activity Toward Chicken α4ß2-Neuronal Nicotinic Acetylcholine Receptors.

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 α4ß2-neuronal nAChRs that were more potent than pHTX.

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster.

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)-ß-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.

Determinants of Subtype-Selectivity of the Anthelmintic Paraherquamide A on Caenorhabditis elegans Nicotinic Acetylcholine Receptors.

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls-AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes.

Neonicotinoid Insecticides: Molecular Targets, Resistance, and Toxicity.

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.

Cofactor-enabled functional expression of fruit fly, honeybee, and bumblebee nicotinic receptors reveals picomolar neonicotinoid actions.

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.

Competitive chrodrimanin B interactions with rat brain GABAA receptors revealed by radioligand binding assays.

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.

Effects of cofactors RIC-3, TMX3 and UNC-50, together with distinct subunit ratios on the agonist actions of imidacloprid on Drosophila melanogaster Dα1/Dß1 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

Insect nicotinic acetylcholine receptors (nAChRs) require cofactors for functional heterologous expression. A previous study revealed that TMX3 was crucial for the functional expression of Drosophila melanogaster Dα1/Dß1 nAChRs in Xenopus laevis oocytes, while UNC-50 and RIC-3 enhanced the acetylcholine (ACh)-induced responses of the nAChRs. However, it is unclear whether the coexpression of UNC-50 and RIC-3 with TMX3 and the subunit stoichiometry affect pharmacology of Dα1/Dß1 nAChRs when expressed in X. laevis oocytes. We have investigated the effects of coexpressing UNC-50 and RIC-3 with TMX3 as well as changing the subunit stoichiometry on the agonist activity of ACh and imidacloprid on the Dα1/Dß1 nAChRs. UNC-50 and RIC-3 hardly affected the agonist affinity of ACh and imidacloprid for the Dα1/Dß1 nAChRs formed by injecting into X. laevis oocytes with an equal amount mixture of the subunit cRNAs, but enhanced current amplitude of the ACh-induced response. Imidacloprid showed higher affinity for the Dß1 subunit-excess Dα1/Dß1 (Dα1/Dß1 = 1/5) nAChRs than the Dα1 subunit-excess Dα1/Dß1 (Dα1/Dß1 = 5/1) nAChRs, suggesting that imidacloprid prefers the Dα1-Dß1 orthosteric site over the Dα1-Dα1 orthosteric site.

Chemical and biological studies of natural and synthetic products for the highly selective control of pest insect species.

Tanacetum cinerariifolium was known to produce pyrethrins, but the mechanism of pyrethrin biosynthesis was largely unclear. The author showed that the nonmevalonate 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)-ß-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 nontarget organisms. The author discovered cofactors 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 crop pests and disease vectors.

A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA-gated chloride channel blockers in the two-spotted spider mite Tetranychus urticae Koch.

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.

Modulation by neonicotinoids of honeybee α1/chicken ß2 hybrid nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

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 ß2 hybrid nAChRs in Xenopus laevis oocytes according to the subunit stoichiometry of (Amα1)3(ß2)2 and (Amα1)2(ß2)3 using voltage-clamp electrophysiology. ACh activated (Amα1)3(ß2)2 and (Amα1)2(ß2)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/ß2 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.

Feeding experiment using uniformly 13C-labeled α-linolenic acid supports the involvement of the decarboxylation mechanism to produce cis-jasmone in Lasiodiplodia theobromae.

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.

Combined effects of mutations in loop C and the loop D-E-G triangle on neonicotinoid interactions with Drosophila Dα1/chicken ß2 hybrid nAChRs.

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 ß2 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.

Action of six pyrethrins purified from the botanical insecticide pyrethrum on cockroach sodium channels expressed in Xenopus oocytes.

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.

Splice Variants of pH-Sensitive Chloride Channel Identify a Key Determinant of Ivermectin Sensitivity in the Larvae of the Silkworm Bombyx mori.

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.

An L319F mutation in transmembrane region 3 (TM3) selectively reduces sensitivity to okaramine B of the Bombyx mori l-glutamate-gated chloride channel.

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 γ-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.

Dr. Kathleen Drew-Baker, "Mother of the Sea", a Manchester scientist celebrated each year for half a century in Japan.

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.

Probing new components (loop G and the α-α interface) of neonicotinoid binding sites on nicotinic acetylcholine receptors.

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 ß1 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.