The effect of toxic pyridine-alkaloid secondary metabolites on the sunbird gut microbiome.

Sunbirds feed on tobacco tree nectar which contains toxic nicotine and anabasine secondary metabolites. Our aim was to understand the effect of nicotine and anabasine on the gut microbiota composition of sunbirds. Sixteen captive sunbirds were randomly assigned to two diets: artificial nectar either with (treatment) or without (control) added nicotine and anabasine. Excreta were collected at 0, 2, 4 and 7 weeks of treatment and samples were processed for bacterial culture and high-throughput amplicon sequencing of the 16S rRNA gene. The gut microbiome diversity of the treated and control birds changed differently along the seven-week experiment. While the diversity decreased in the control group along the first three samplings (0, 2 and 4 weeks), it increased in the treatment group. The microbiota composition analyses demonstrated that a diet with nicotine and anabasine, significantly changed the birds' gut microbiota composition compared to the control birds. The abundance of nicotine- and anabasine- degrading bacteria in the excreta of the treated birds, was significantly higher after four and seven weeks compared to the control group. Furthermore, analysis of culturable isolates, including Lactococcus, showed that sunbirds' gut-associated bacteria were capable of degrading nicotine and anabasine, consistent with their hypothesised role as detoxifying and nutritional symbionts.

Increased Leaf Nicotine Content by Targeting Transcription Factor Gene Expression in Commercial Flue-Cured Tobacco (Nicotiana tabacum L.).

Nicotine, the most abundant pyridine alkaloid in cultivated tobacco (Nicotiana tabacum L.), is a potent inhibitor of insect and animal herbivory and a neurostimulator of human brain function. Nicotine biosynthesis is controlled developmentally and can be induced by abiotic and biotic stressors via a jasmonic acid (JA)-mediated signal transduction mechanism involving members of the APETALA 2/ethylene-responsive factor (AP2/ERF) and basic helix-loop-helix (bHLH) transcription factor (TF) families. AP2/ERF and bHLH TFs work combinatorically to control nicotine biosynthesis and its subsequent accumulation in tobacco leaves. Here, we demonstrate that overexpression of the tobacco NtERF32, NtERF221/ORC1, and NtMYC2a TFs leads to significant increases in nicotine accumulation in T2 transgenic K326 tobacco plants before topping. Up to 9-fold higher nicotine production was achieved in transgenics overexpressing NtERF221/ORC1 under the control of a constitutive GmUBI3 gene promoter compared to wild-type plants. The constitutive 2XCaMV35S promoter and a novel JA-inducible 4XGAG promoter were less effective in driving high-level nicotine formation. Methyljasmonic acid (MeJA) treatment further elevated nicotine production in all transgenic lines. Our results show that targeted manipulation of NtERF221/ORC1 is an effective strategy for elevating leaf nicotine levels in commercial tobacco for use in the preparation of reduced risk tobacco products for smoking replacement therapeutics.

Context-dependent medicinal effects of anabasine and infection-dependent toxicity in bumble bees.

BACKGROUND: Floral phytochemicals are ubiquitous in nature, and can function both as antimicrobials and as insecticides. Although many phytochemicals act as toxins and deterrents to consumers, the same chemicals may counteract disease and be preferred by infected individuals. The roles of nectar and pollen phytochemicals in pollinator ecology and conservation are complex, with evidence for both toxicity and medicinal effects against parasites. However, it remains unclear how consistent the effects of phytochemicals are across different parasite lineages and environmental conditions, and whether pollinators actively self-medicate with these compounds when infected. APPROACH: Here, we test effects of the nectar alkaloid anabasine, found in Nicotiana, on infection intensity, dietary preference, and survival and performance of bumble bees (Bombus impatiens). We examined variation in the effects of anabasine on infection with different lineages of the intestinal parasite Crithidia under pollen-fed and pollen-starved conditions. RESULTS: We found that anabasine did not reduce infection intensity in individual bees infected with any of four Crithidia lineages that were tested in parallel, nor did anabasine reduce infection intensity in microcolonies of queenless workers. In addition, neither anabasine nor its isomer, nicotine, was preferred by infected bees in choice experiments, and infected bees consumed less anabasine than did uninfected bees under no-choice conditions. Furthermore, anabasine exacerbated the negative effects of infection on bee survival and microcolony performance. Anabasine reduced infection in only one experiment, in which bees were deprived of pollen and post-pupal contact with nestmates. In this experiment, anabasine had antiparasitic effects in bees from only two of four colonies, and infected bees exhibited reduced-rather than increased-phytochemical consumption relative to uninfected bees. CONCLUSIONS: Variation in the effect of anabasine on infection suggests potential modulation of tritrophic interactions by both host genotype and environmental variables. Overall, our results demonstrate that Bombus impatiens prefer diets without nicotine and anabasine, and suggest that the medicinal effects and toxicity of anabasine may be context dependent. Future research should identify the specific environmental and genotypic factors that determine whether nectar phytochemicals have medicinal or deleterious effects on pollinators.

The effects of neonicotinoid exposure on embryonic development and organ mass in northern bobwhite quail (Colinus virginianus).

Since their emergence in the early 1990s, neonicotinoid use has increased exponentially to make them the world's most prevalent insecticides. Although there has been considerable research concerning the lethality of neonicotinoids, their sub-lethal and developmental effects are still being explored, especially with regard to non-mammalian species. The goal of this research was to investigate the effects of the neonicotinoid imidacloprid on the morphological and physiological development of northern bobwhite quail (Colinus virginianus). Bobwhite eggs (n=390) were injected with imidacloprid concentrations of 0 (sham), 10, 50, 100, and 150mg/kg of egg mass, which was administered at day 0 (pre-incubation), 3, 6, 9, or 12 of growth. Embryos were dissected, weighed, staged, and examined for any overt structural deformities after 19days of incubation. The mass of the embryonic heart, liver, lungs and kidneys was also recorded. The majority of treatments produced no discernible differences in embryo morphology; however, in some instances, embryos were subject to increased frequency of anatomical deformity and altered organ masses. Some impacts were more pronounced in specific dosing periods, implying that there may be critical windows of development when embryos are more susceptible to neonicotinoid exposure. This investigation suggests that imidacloprid has the potential to impact bobwhite quail embryonic development and chick survival.

Monitoring of neonicotinoid pesticides in beekeeping.

The decline of pollinating species is correlated to the extensive use of neonicotinoids against pest insects for crop protection. In this study, the concentrations of neonicotinoid insecticides were determined in honeybees, honeycomb and honey samples, collected in Spring 2015 (blooming period) from different areas in Sicily (IT), to carry out an evaluation of bees products' safety and an overview of neonicotinoid contamination in beekeeping. The results obtained showed only the presence of clothianidin in bee samples and these concentrations don't represent a risk for bees' vitality and safety. The absence of residue in all honey samples, instead, showed the quality of bee products.

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.

Molecular Effects of Neonicotinoids in Honey Bees (Apis mellifera).

Neonicotinoids are implicated in the decline of bee populations. As agonists of nicotinic acetylcholine receptors, they disturb acetylcholine receptor signaling leading to neurotoxicity. Several behavioral studies showed the link between neonicotinoid exposure and adverse effects on foraging activity and reproduction. However, molecular effects underlying these effects are poorly understood. Here we elucidated molecular effects at environmental realistic levels of three neonicotinoids and nicotine, and compared laboratory studies to field exposures with acetamiprid. We assessed transcriptional alterations of eight selected genes in caged honey bees exposed to different concentrations of the neonicotinoids acetamiprid, clothianidin, imidacloporid, and thiamethoxam, as well as nicotine. We determined transcripts of several targets, including nicotinic acetylcholine receptor α 1 and α 2 subunit, the multifunctional gene vitellogenin, immune system genes apidaecin and defensin-1, stress-related gene catalase and two genes linked to memory formation, pka and creb. Vitellogenin showed a strong increase upon neonicotinoid exposures in the laboratory and field, while creb and pka transcripts were down-regulated. The induction of vitellogenin suggests adverse effects on foraging activity, whereas creb and pka down-regulation may be implicated in decreased long-term memory formation. Transcriptional alterations occurred at environmental concentrations and provide an explanation for the molecular basis of observed adverse effects of neonicotinoids to bees.

Neonicotinoid Residues in Wildflowers, a Potential Route of Chronic Exposure for Bees.

In recent years, an intense debate about the environmental risks posed by neonicotinoids, a group of widely used, neurotoxic insecticides, has been joined. When these systemic compounds are applied to seeds, low concentrations are subsequently found in the nectar and pollen of the crop, which are then collected and consumed by bees. Here we demonstrate that the current focus on exposure to pesticides via the crop overlooks an important factor: throughout spring and summer, mixtures of neonicotinoids are also found in the pollen and nectar of wildflowers growing in arable field margins, at concentrations that are sometimes even higher than those found in the crop. Indeed, the large majority (97%) of neonicotinoids brought back in pollen to honey bee hives in arable landscapes was from wildflowers, not crops. Both previous and ongoing field studies have been based on the premise that exposure to neonicotinoids would occur only during the blooming period of flowering crops and that it may be diluted by bees also foraging on untreated wildflowers. Here, we show that exposure is likely to be higher and more prolonged than currently recognized because of widespread contamination of wild plants growing near treated crops.

An Observational Study of Honey Bee Colony Winter Losses and Their Association with Varroa destructor, Neonicotinoids and Other Risk Factors.

This article presents results of an analysis of honey bee losses over the winter of 2011-2012 in the Netherlands, from a sample of 86 colonies, located at 43 apiaries. The apiaries were selected using spatially stratified random sampling. Colony winter loss data were collected and related to various measures of colony strength recorded in summer, as well as data from laboratory analysis of sample material taken from two selected colonies in each of the 43 apiaries. The logistic regression model which best explained the risk of winter loss included, in order of statistical importance, the variables (1) Varroa destructor mite infestation rate in October 2011, (2) presence of the cyano-substituted neonicotinoids acetamiprid or thiacloprid in the first 2 weeks of August 2011 in at least one of the honey bee matrices honey, bees or bee bread (pollen), (3) presence of Brassica napus (oilseed rape) or Sinapis arvensis (wild mustard) pollen in bee bread in early August 2011, and (4) a measure of the unexplained winter losses for the postal code area where the colonies were located, obtained from a different dataset. We consider in the discussion that reduced opportunities for foraging in July and August because of bad weather may have added substantially to the adverse effects of acetamiprid and thiacloprid. A novel feature of this work is its use of postal code random effects from two other independent datasets collected in the annual national monitoring by questionnaires of winter losses of honey bees in the Netherlands. These were used to plan the sample selection and also in the model fitting of the data in this study. It should however be noted that the results of the present pilot study are based on limited data, which may consequently reveal strong factors but fail to demonstrate possible interaction effects.

The effect of intermittent dosing of Nicotiana glauca on teratogenesis in goats.

Sustained inhibition of fetal movement in livestock species, induced by several poisonous plants, can result in numerous skeletal-contracture malformations. Lupines are responsible for a condition in cattle referred to as "crooked calf syndrome" that occurs when pregnant cattle graze teratogenic lupines. Similar malformations are also seen in animals poisoned by Conium maculatum (coniine) and Nicotiana glauca (anabasine). A proposed management strategy to limit these types of birth defects includes utilizing an intermittent grazing schedule to allow short durations of grazing lupine-infested areas interrupted by movement to a lupine-free pasture. The objective of this study was to use a goat model to determine if an intermittent schedule of five continuous days on treatment followed by two days off treatment would be sufficient to decrease, or prevent, the incidence of anabasine-induced malformations. The data from this study suggest that, for N. glauca in goats, the intermittent grazing program of five days exposure with two days of non-exposure is insufficient to prevent significant skeletal malformations from occurring. However, this study did demonstrate an inverse relationship between the amount of serum anabasine in the dam and the extent of fetal movement.

Studies on the teratogenicity of anabasine in a rat model.

A number of plant toxins have been shown to be teratogenic to livestock. The teratogenic action of some of these alkaloids is mediated by nicotinic acetylcholine receptors (nAChR). However, for many of these alkaloids it is difficult to obtain sufficient quantities of individual alkaloids to perform teratology studies in livestock species. Therefore the objective of this study was to determine if a rat model can be utilized to characterize the teratogenic nature of individual plant toxins that are nAChR agonists. In this study, we evaluated the teratogenicity of anabasine by feeding pregnant rats anabasine-containing rodent chow from gestational day (GD) 6-21. On GD21, the dams were euthanized and the gravid uteri were removed. The gravid uteri and individual pups were weighed. The pups were evaluated for bone malformations including cleft palate and scoliosis. Overall, the results of this study suggest that the rat is not a good model to study the teratogenicity of plant toxins that are nAChR agonists. It is possible that in the rat model, anabasine administered orally via the chow may not result in sufficient reduction in fetal movement to cause the significant malformations observed in livestock species.

A restatement of the natural science evidence base concerning neonicotinoid insecticides and insect pollinators.

There is evidence that in Europe and North America many species of pollinators are in decline, both in abundance and distribution. Although there is a long list of potential causes of this decline, there is concern that neonicotinoid insecticides, in particular through their use as seed treatments are, at least in part, responsible. This paper describes a project that set out to summarize the natural science evidence base relevant to neonicotinoid insecticides and insect pollinators in as policy-neutral terms as possible. A series of evidence statements are listed and categorized according to the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Honey bees, neonicotinoids and bee incident reports: the Canadian situation.

BACKGROUND: Neonicotinoid insecticides have been the target of much scrutiny as possible causes of recent declines observed in pollinator populations. Although neonicotinoids have been implicated in honey bee pesticide incidents, there has been little examination of incident report data. Here we summarize honey bee incident report data obtained from the Canadian Pest Management Regulatory Agency (PMRA). RESULTS: In Canada, there were very few honey bee incidents reported in 2007-2011 and data were not collected prior to 2007. In 2012, a significant number of incidents were reported in the province of Ontario, where exposure to neonicotinoid dust during planting of corn was suspected to have caused the incident in up to 70% of cases. Most of these incidents were classified as 'minor' by the PMRA, and only six cases were considered 'moderate' or 'major'. In that same year, there were over three times as many moderate or major incidents due to older non-neonicotinoid pesticides, involving numbers of hives or bees far greater than the number of moderate or major incidents suspected to be due to neonicotinoid poisoning. CONCLUSIONS: These data emphasize that, while exposure of honey bees to neonicotinoid-contaminated dust during corn planting needs to be mitigated, other pesticides also pose a risk.

Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees.

Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

The role of the α7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllycaconitine and anabasine.

The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. However, recent research demonstrated that there was no difference in the acute lethality (LD50 ) to MLA in mice lacking the α7 nAChR subunit compared with wild-type mice. The objective of this study was to determine if the α7 nAChR subunit plays a role in motor coordination deficiencies that result from exposure to nAChR antagonists and agonists. We compared the motor function and coordination in wild-type mice to mice lacking the α7 subunit of the nAChR, after treating them with a non-lethal dose of MLA or anabasine, using the following tests: balance beam, grip strength, rotarod, open field and tremor monitor. Analysis of the data indicated that overall there was no difference between the wild-type and knockout mice (P = 0.39 for grip strength; P = 0.21 for rotarod; P = 0.41 for balance beam; P = 0.22 for open field; and P = 0.62 for tremors). Thus results from this study suggest that α7 nAChR does not play an integral role in the acute effects of MLA or anabasine on motor function/coordination. Consequently other subunits of nAChRs found in the neuromuscular junction are likely the primary target for MLA and anabasine resulting in motor coordination deficiencies and acute toxicosis.

Sublethal effects of paichongding on Nilaparvata lugens (Homoptera: Delphacidae).

Nilaparvata lugens (Stål) is a major rice pest in Asia Paichongding is a novel neonicotinoid insecticide developed in 2008. The effects of this insecticide on life-table parameters and wing formation of N. lugens were examined in the laboratory. The results showed that paichongding could significantly reduce the fecundity of N. lugens, demonstrating further activity against this pest in addition to direct toxicity. The fecundity of N. lugens treated with LC20, LC30, LC40, and LC50 of paichongding were 87.44, 79.63, 63.31, and 52.66% of the control, respectively. The relative fitness values were 0.55, 0.41, 0.21, and 0.09, respectively, for LC20, LC30, LC40, and LC50 paichongding treatments. Sublethal concentrations of paichongding showed significant induction of macropterous offspring, which would be one kind of coping mechanism of N. lugens response to insecticide stress.

Survey of neonicotinoids and fipronil in corn seeds for agriculture.

Recently, legislative decisions withdrew or temporarily suspended the use of neonicotinoids and fipronil as seeds tanning in many countries because of their endocrine-disrupting activity imputable to the bees' toxicity. In this study, the occurrence of acetamiprid, fipronil, clothianidin, flonicamid, imidacloprid, nitenpyram, thiacloprid and thiamethoxam was detected in 66 samples of commercial treated corn seeds, collected in the Italian market in the frame of ministerial institutional quality control activity. Because of the lack of a validated analytical protocol for neonicotinoid detection in seeds, a routinely suitable liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analytical method was developed and statistically validated on fortified corn seeds. Survey results demonstrated that 88% of the investigated seed samples showed the presence of residues of clothianidin, fipronil, thiamethoxam and thiacloprid, either individually or simultaneously, with values that ranged from about 0.002 to 20 mg kg(-1), which evidenced the alarming illicit use of these pesticides in seed treatments.