Subchronic effects of plant alkaloids on anxiety-like behavior in zebrafish.

Zebrafish provide a valuable emerging complementary model for neurobehavioral research. They offer a powerful way to screen for the potential therapeutic effects of neuroactive drugs. A variety of behavioral tests for zebrafish have been developed and validated for assessing neurobehavioral function. The novel tank diving test is a straightforward, reproducible way of measuring anxiety-like behavior in zebrafish. When introduced into a novel tank, zebrafish normally dive to the bottom of the tank and then gradually explore the higher levels of the water column as time progresses. Buspirone is an effective anxiolytic drug in humans, which has been found, with acute administration, to reduce this anxiety-like response in zebrafish. The current study used the zebrafish model to evaluate the potential anxiolytic effects of alkaloids, commonly found in Solanaceae plants, with known neuropharmacology relevant to mood regulation. In line with previous findings, acute treatment with anxiolytic positive controls buspirone and the plant alkaloid nicotine reduced the anxiety-like diving response in the zebrafish novel tank diving test. Further, both buspirone and nicotine continued to produce anxiolytic-like effects in zebrafish after 5 days of exposure. In the same treatment paradigm, the effects of five other alkaloids-cotinine, anatabine, anabasine, harmane, and norharmane-were investigated. Cotinine, the major metabolite of nicotine, also caused anxiolytic-like effects, albeit at a dose higher than the effective dose of nicotine. Nicotine's anxiolytic-like effect was not shared by the other nicotinic alkaloids, anabasine and anatabine, or by the naturally present monoamine oxidase inhibitors harmane and norharmane. We conclude that nicotine uniquely induces anxiolytic-like effects after acute and subchronic treatment in zebrafish. The zebrafish model with the novel tank diving test could be a useful complement to rodent models for screening candidate compounds for anxiolytic effects in nonclinical studies.

Spleen is not required for therapeutic effects of 4OH-GTS-21, a selective α7 nAChR agonist, in the sub-acute phase of ischemic stroke in rats.

Acute ischemic stroke (AIS) causes both central and peripheral inflammation, while activation of α7 nicotinic acetylcholine receptors (nAChRs) provides both central and peripheral anti-inflammatory and anti-apoptotic effects. Here, we provide evidence that 4OH-GTS-21, a selective α7 agonist, produces its therapeutic effects via primarily central sites of action because 4OH-GTS-21 was found equally effective in splenectomized and non-spenectomized rats in the sub-acute phase of ischemic stroke (≤1 week). However, the spleen may boost the therapeutic efficacy of 4OH-GTS-21 in certain behavioral tasks as our data also indicated. In our tests, AIS was modeled by transient middle cerebral artery occlusion (tMCAO). Splenectomy was done 2 weeks before tMCAO. We determined that: 1) Daily 4OH-GTS-21 treatments for 7 days after tMCAO significantly reduced neurological deficits and brain injury in both splenectomized and non-spelenectomized rats demonstrating that the spleen is not required for therapeutic benefits of 4OH-GTS-21; 2) The effects of 4OH-GTS-21 in the adhesive sticker removal test were significantly weaker in splenectomized animals suggesting that the spleen boosts the efficacy of 4OH-GTS-21 in the first week after tMCAO; and 3) Ischemic brain injury was not significantly affected by splenectomy in both vehicle-treated and 4OH-GTS-21-treated animals. These data support the hypothesis that the therapeutic efficacy of sub-chronic (≤1 week) 4OH-GTS-21 primarily originates from central sites of action. These results validate brain availability as a critical factor for developing novel α7 ligands for AIS.

A Pharmacological Comparison of Two Isomeric Nicotinic Receptor Agonists: The Marine Toxin Isoanatabine and the Tobacco Alkaloid Anatabine.

Many organisms possess "secondary" compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other "minor" compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing anabasine analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4ß2 nicotinic acetylcholine receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand binding experiments revealed similar α4ß2 nAChR binding affinities for the isoanatabines, but R-anatabine affinity was twice that of S-anatabine. While the two anatabines and S-isoanatabine were highly efficacious agonists at α7 nAChRs, R-isoanatabine was only a weak partial agonist. The four compounds share an ability to stimulate both α4ß2 and α7 nAChRs, a property that may be useful in developing more efficacious drugs to treat neurodegenerative and other medical disorders.

Investigation of the Possible Pharmacologically Active Forms of the Nicotinic Acetylcholine Receptor Agonist Anabaseine.

Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [3H]-methylcarbamylcholine binding at rat brain α4ß2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cyclic imine analog 2,3'-bipyridyl and the open-chain ammonium-ketone analog 5-methylamino-1-(3-pyridyl)-1-pentanone (MAPP), displayed ≤1% of the activity predicted if the one form was solely active. The lower potency of weakly basic 2,3'-bipyridyl can be explained by the presence of a small concentration of its monocationic form. Since the open chain ammonium-ketone monocationic form of anabaseine has some structural similarity to the neurotransmitter GABA, we also tested the ability of anabaseine and its 1,2-dehydropyrrolidinyl analog myosmine to activate a mammalian GABAA receptor, but no activity was detected. We conclude that the monocationic cyclic iminium is the form which avidly binds and activates vertebrate nAChRs.

Complete inhibition of fetal movement in the day 40 pregnant goat model by the piperidine alkaloid anabasine but not related alkaloids.

Four chemically similar alkaloids, anabasine, anabaseine, epibatidine and dimethylphenylpiperazinium (DMPP), are potent nicotinic acetylcholine receptor agonists of fetal muscle nicotinic acetylcholine receptors in human TE-671 cells. Based on results with these cells, we hypothesized that the alkaloids would completely inhibit ultrasound-monitored fetal movement in a goat model. Different, single doses of anabasine, anabaseine, epibatidine, DMPP, or saline control were administered I.V. to pregnant goats on day 40 of gestation and the number of fetal movements per 5 min sample was measured by ultrasound at times 0, 0.5, 1, 2, 4 and 8 h. The differences among does in fetal movements were more consistent at dosing and following recovery for doses of anabasine above 0.125 mg/kg compared to the other compounds and dosages. Anabasine actions were dose-dependent with an IC50 value of ∼0.1 mg/kg, and, at a dose of 0.8 mg/kg, completely inhibited fetal movement for 1.5 h after dosing. Anabaseine, epibatidine, and DMPP failed to completely inhibit fetal movement in day 40 pregnant goats at doses predicted to be effective. These results suggest that while experiments with TE-671 cells provide valuable information and predictions of the actions of plant alkaloids on fetal movement, in vivo experiments are still required in order to determine the ability of an alkaloid to inhibit fetal movement in livestock species. Moreover, other pharmacological properties such as receptor differences between mammalian species and differences in the pharmacokinetic properties of the alkaloids also are likely to weaken teratologic predictions based solely on the in vitro data.

Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

Arthropod Pest Control for UK Oilseed Rape - Comparing Insecticide Efficacies, Side Effects and Alternatives.

Oilseed rape (Brassica napus) is an important combinable break crop in the UK, which is largely protected from arthropod pests by insecticidal chemicals. Despite ongoing debate regarding the use of neonicotinoids, the dominant seed treatment ingredients used for this crop, there is little publicly available data comparing the efficacy of insecticides in controlling key arthropod pests or comparing the impacts on non-target species and the wider environment. To provide an insight into these matters, a UK-wide expert survey targeting agronomists and entomologists was conducted from March to June 2015. Based on the opinions of 90 respondents, an average of 20% yield loss caused by the key arthropod pests was expected to have occurred in the absence of insecticide treatments. Relatively older chemical groups were perceived to have lower efficacy for target pests than newer ones, partly due to the development of insecticide resistance. Without neonicotinoid seed treatments, a lack of good control for cabbage stem flea beetle was perceived. Wide spectrum foliar insecticide sprays were perceived to have significantly greater negative impacts than seed treatments on users' health, natural enemies, pollinators, soil and water, and many foliar active ingredients have had potential risks for non-target arthropod species in UK oilseed rape fields for the past 25 years. Overall, 72% of respondents opposed the neonicotinoid restriction, while 10% supported it. Opposition and support of the restriction were largely based on concerns for pollinators and the wider environment, highlighting the uncertainty over the side effects of neonicotinoid use. More people from the government and research institutes leaned towards neutrality over the issue, compared to those directly involved in growing the crop. Neonicotinoid restriction was expected to result in greater effort and expenditure on pest control and lower production (0-1 t/ha less). Alternatives for future oilseed rape protection were then discussed.

Pleiotropic Effects of Loss of the Dα1 Subunit in Drosophila melanogaster: Implications for Insecticide Resistance.

Nicotinic acetylcholine receptors (nAChRs) are a highly conserved gene family that form pentameric receptors involved in fast excitatory synaptic neurotransmission. The specific roles individual nAChR subunits perform in Drosophila melanogaster and other insects are relatively uncharacterized. Of the 10 D. melanogaster nAChR subunits, only three have described roles in behavioral pathways; Dα3 and Dα4 in sleep, and Dα7 in the escape response. Other subunits have been associated with resistance to several classes of insecticides. In particular, our previous work has demonstrated that an allele of the Dα1 subunit is associated with resistance to neonicotinoid insecticides. We used ends-out gene targeting to create a knockout of the Dα1 gene to facilitate phenotypic analysis in a controlled genetic background. To our knowledge, this is the first report of a native function for any nAChR subunits known to be targeted by insecticides. Loss of Dα1 function was associated with changes in courtship, sleep, longevity, and insecticide resistance. While acetylcholine signaling had previously been linked with mating behavior and reproduction in D. melanogaster, no specific nAChR subunit had been directly implicated. The role of Dα1 in a number of behavioral phenotypes highlights the importance of understanding the biological roles of nAChRs and points to the fitness cost that may be associated with neonicotinoid resistance.

(S)-5-ethynyl-anabasine, a novel compound, is a more potent agonist than other nicotine alkaloids on the nematode Asu-ACR-16 receptor.

Nematode parasites infect ∼2 billion people world-wide. Infections are treated and prevented by anthelmintic drugs, some of which act on nicotinic acetylcholine receptors (nAChRs). There is an unmet need for novel therapeutic agents because of concerns about the development of resistance. We have selected Asu-ACR-16 from a significant nematode parasite genus, Ascaris suum, as a pharmaceutical target and nicotine as our basic moiety (EC50 6.21 ± 0.56 µM, Imax 82.39 ± 2.52%) to facilitate the development of more effective anthelmintics. We expressed Asu-ACR-16 in Xenopus oocytes and used two-electrode voltage clamp electrophysiology to determine agonist concentration-current-response relationships and determine the potencies (EC50s) of the agonists. Here, we describe the synthesis of a novel agonist, (S)-5-ethynyl-anabasine, and show that it is more potent (EC50 0.14 ± 0.01 µM) than other nicotine alkaloids on Asu-ACR-16. Agonists acting on ACR-16 receptors have the potential to circumvent drug resistance to anthelmintics, like levamisole, that do not act on the ACR-16 receptors.

Bumble bee parasite strains vary in resistance to phytochemicals.

Nectar and pollen contain diverse phytochemicals that can reduce disease in pollinators. However, prior studies showed variable effects of nectar chemicals on infection, which could reflect variable phytochemical resistance among parasite strains. Inter-strain variation in resistance could influence evolutionary interactions between plants, pollinators, and pollinator disease, but testing direct effects of phytochemicals on parasites requires elimination of variation between bees. Using cell cultures of the bumble bee parasite Crithidia bombi, we determined (1) growth-inhibiting effects of nine floral phytochemicals and (2) variation in phytochemical resistance among four parasite strains. C. bombi growth was unaffected by naturally occurring concentrations of the known antitrypanosomal phenolics gallic acid, caffeic acid, and chlorogenic acid. However, C. bombi growth was inhibited by anabasine, eugenol, and thymol. Strains varied >3-fold in phytochemical resistance, suggesting that selection for phytochemical resistance could drive parasite evolution. Inhibitory concentrations of thymol (4.53-22.2 ppm) were similar to concentrations in Thymus vulgaris nectar (mean 5.2 ppm). Exposure of C. bombi to naturally occurring levels of phytochemicals-either within bees or during parasite transmission via flowers-could influence infection in nature. Flowers that produce antiparasitic phytochemicals, including thymol, could potentially reduce infection in Bombus populations, thereby counteracting a possible contributor to pollinator decline.

[EFFECT OF MEDIATOR-TYPE DRUGS ON HUMAN PSYCHOPHYSIOLOGICAL STATUS DURING MODEL OPERATOR ACTIVITY].

In a placebo-controlled study, changes in psychophysiological status of operators (38 healthy male volunteers aged 23-35 years) performing 4-hour model operator activity were evaluated after a single oral administration of typical representatives of the different classes of drugs (haloperidol, proroxan, yohimbine hydrochloride, propranolol, mesocarb, isoprenaline, Belladonna extract, anabasine hydrochloride, valproate sodium, and phenazepam), which are used for the treatment, rehabilitation and prophylaxis of common diseases. It was found that all the drugs modified to a greater or lesser extent some components of the model operator activity. Isoprenaline and phenazepam had the most negative effect on the psychophysiological indicators and quality of the modeled operator activity. The results should be considered before administration of such drugs to working operators.

Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids.

Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic.

Specific Synergist for Neonicotinoid Insecticides: IPPA08, a cis-Neonicotinoid Compound with a Unique Oxabridged Substructure.

Insecticide synergists are key components to increase the control efficacy and reduce active ingredient use. Here, we describe a novel insecticide synergist with activity specific for insecticidal neonicotinoids. The synergist IPPA08, a cis configuration neonicotinoid compound with a unique oxabridged substructure, could increase the toxicity of most neonicotinoid insecticides belonging to the Insecticide Resistance Action Committee (IRAC) 4A subgroup against a range of insect species, although IPPA08 itself was almost inactive to insects at synergistic concentrations. Unfortunately, similar effects were observed on the honey bee (Apis mellifera) and the brown planthopper (Nilaparvata lugens), resistant to imidacloprid. IPPA08 did not show any effects on toxicity of insecticides with different targets, which made us define it as a neonicotinoid-specific synergist. Unlike most insecticide synergists, by inhibition of activities of detoxification enzymes, IPPA08 showed no effects on enzyme activities. The results revealed that IPPA08 worked as a synergist through a distinct way. Although the modulating insect nicotinic acetylcholine receptors (nAChRs, targets of neonicotinoid insecticides) were supposed as a possible mode of action for IPPA08 as a neonicotinoid-specific synergist, direct evidence is needed in further studies. In insect pest control, IPPA08 acts as a target synergist to increase neonicotinoid toxicity and reduce the amount of neonicotinoid used. Combinations of IPPA08 and insecticidal neonicotinoids may be developed into new insecticide formulations. In summary, combining an active ingredient with a "custom" synergist appears to be a very promising approach for the development of effective new insecticide products.

Nicotinic effects of tobacco smoke constituents in nonhuman primates.

RATIONALE: Recent studies in rodents suggest that non-nicotine constituents of tobacco smoke (e.g., minor tobacco alkaloids) may promote tobacco consumption-either through their own pharmacological effects or by augmenting the effects of nicotine. However, there is scant information on the behavioral pharmacology of minor tobacco alkaloids in primate species. OBJECTIVE: The present studies were conducted to determine whether the minor tobacco alkaloids nornicotine, anabasine, anatabine, myosmine, and cotinine exhibit nicotine-like behavioral effects in squirrel monkeys. METHODS: Initial experiments were conducted to determine the effects of nicotine (0.032-1.0 mg/kg) and the minor tobacco alkaloids nornicotine (1-1.8 mg/kg), anabasine (0.1-1.0 mg/kg), anatabine (10-32 mg/kg), myosmine (0.32-1.8 mg/kg), and cotinine (10-180 mg/kg) on food-maintained performance (n = 4). Next, the ability of tobacco alkaloids to substitute for the α4ß2-selective nicotinic agonist (+)-epibatidine in drug discrimination experiments was evaluated in a separate group of monkeys (n = 4). RESULTS: Results show that nicotine and each minor tobacco alkaloid except cotinine (a) produced dose-related decreases in food-maintained responding; (b) substituted for (+)-epibatidine and, in additional experiments, produced additive effects when combined with nicotine; (c) induced emesis or tremor at doses that reduced food-maintained responding and had (+)-epibatidine-like discriminative-stimulus effects; and (d) based on correlation with reported receptor binding affinities, likely produced their behavioral effects through α4ß2 receptor mechanisms. CONCLUSION: Selected minor tobacco alkaloids have nicotinic-like effects that may contribute to tobacco consumption and addiction.

Induced thiacloprid insensitivity in honeybees (Apis mellifera L.) is associated with up-regulation of detoxification genes.

Honey bees, Apis mellifera, are markedly less sensitive to neonicotinoid insecticides containing a cyanoimino pharmacophore than to those with a nitroimino group. Although previous work has suggested that this results from enhanced metabolism of the former by detoxification enzymes, the specific enzyme(s) involved remain to be characterized. In this work, a pretreatment of honey bees with a sublethal dose of thiacloprid resulted in induced insensitivity to the same compound immediately following thiacloprid feeding. A longer pretreatment time resulted in no, or increased, sensitivity. Transcriptome profiling, using microarrays, identified a number of genes encoding detoxification enzymes that were over-expressed significantly in insecticide-treated bees compared with untreated controls. These included five P450s, CYP6BE1, CYP305D1, CYP6AS5, CYP315A1, CYP301A1, and a carboxyl/cholinesterase (CCE) CCE8. Four of these P450s were functionally expressed in Escherichia coli and their ability to metabolize thiacloprid examined by liquid chromatography-mass spectrometry (LC-MS) analysis.

Modification of the anabaseine pyridine nucleus allows achieving binding and functional selectivity for the α3ß4 nicotinic acetylcholine receptor subtype.

We report the design, synthesis and pharmacological screening of a group of analogues of anabaseine 2, a naturally occurring unselective nicotinic agonist. The novel nAChR ligands 5-15 were planned following a molecular modeling analysis which suggested the replacement of the pyridine ring of 2 with a 3-substituted benzene ring as a means to gain selectivity for the α3ß4 nAChR subtype. Overall, from binding experiments, the synthesized compounds showed high values of α3ß4 affinity and α3ß4 vs α4ß2 selectivity, although they poorly discriminated the homomeric α7 subtype. The three analogues 6, 12 and 13 were also evaluated in electrophysiological assays, and 12 [6-(3-iodophenyl)-2,3,4,5-tetrahydropyridine] emerged as a rather interesting nicotinic ligand. Indeed, in addition to a noteworthy affinity (Ki = 4.7 nM) for the α3ß4 subtype and to an excellent α3ß4 vs α4ß2 subtype selectivity (806-fold), compound 12 selectively activated the α3ß4 nAChR (EC50 = 7.4 µM) while eliciting a negligible response at the α7 subtype and no effect at the α4ß2 subtype.

Effect of α7n-Acetylcholine Receptor Activation and Antibodies to TNF-α on Mortality of Mice and Concentration of Proinflammatory Cytokines During Early Stage of Sepsis.

Experiments on random-bred albino mice showed that activation α7n-acetylcholine receptors with anabasine (0.5 LD50) and the use of antibodies to TNF-α (1 mg/kg) 2 h before sepsis modeling significantly reduces mortality of mice from experimental sepsis (intraperitoneal injection of E. coli) due to a decrease in the blood concentration of proinflammatory cytokines TNF-α, IL-1ß, and IL-6. After combined administration of anti-TNF-α antibodies and anabasine, an additive effect was observed.

Low doses of neonicotinoid pesticides in food rewards impair short-term olfactory memory in foraging-age honeybees.

Neonicotinoids are often applied as systemic seed treatments to crops and have reported negative impact on pollinators when they appear in floral nectar and pollen. Recently, we found that bees in a two-choice assay prefer to consume solutions containing field-relevant doses of the neonicotinoid pesticides, imidacloprid (IMD) and thiamethoxam (TMX), to sucrose alone. This suggests that neonicotinoids enhance the rewarding properties of sucrose and that low, acute doses could improve learning and memory in bees. To test this, we trained foraging-age honeybees to learn to associate floral scent with a reward containing nectar-relevant concentrations of IMD and TMX and tested their short (STM) and long-term (LTM) olfactory memories. Contrary to our predictions, we found that none of the solutions enhanced the rate of olfactory learning and some of them impaired it. In particular, the effect of 10 nM IMD was observed by the second conditioning trial and persisted 24 h later. In most other groups, exposure to IMD and TMX affected STM but not LTM. Our data show that negative impacts of low doses of IMD and TMX do not require long-term exposure and suggest that impacts of neonicotinoids on olfaction are greater than their effects on rewarding memories.

Neonicotinoid insecticide interact with honeybee odorant-binding protein: Implication for olfactory dysfunction.

The decline of bee population has caused great concern in recent years. A noticeable factor points to the neonicotinoid insecticides, which remain in the nectar and pollen of plants and impair the olfactory cognition of foraging bees. However, it remains elusive that if and how neonicotinoid insecticides interact with the olfactory system of bees. Herein, we studied the binding interaction between neonicotinoid imidacloprid and ASP2, one odorant-binding protein in eastern bees, Apis cerana, by multispectroscopic methods. The results indicate that imidacloprid significantly quenched the intrinsic fluorescence of ASP2 as the static quenching mode, and expanded the conformation of ASP2 measured by the circular dichroism (CD) spectra. The acting force is mainly driven by hydrophobic force based on thermodynamic analysis. Docking analysis predicts a formation of a hydrogen bond, while the corresponding site-directed mutagenesis indicated that the hydrogen bond is not main force here. Moreover, imidacloprid with a sublethal dose (0.8ng/bee) clearly decreased the binding affinity of ASP2 to a floral volatile, ß-ionone, which had been identified to strongly bind with the wild ASP2 before. This study may benefit to evaluate the effect of neonicotinoid insecticides on the olfactory cognitive behavior of bees involved in the crops pollination.

Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing.

While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥ 3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. To our knowledge, this is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.