Troy D. Anderson

Risks of neonicotinoid insecticides to honeybees

The European honeybee, Apis mellifera, is an important pollinator of agricultural crops. Since 2006, when unexpectedly high colony losses were first reported, articles have proliferated in the popular press suggesting a range of possible causes and raising alarm over the general decline of bees. Suggested causes include pesticides, genetically modified crops, habitat fragmentation, and introduced diseases and parasites. Scientists have concluded that multiple factors in various combinations—including mites, fungi, viruses, and pesticides, as well as other factors such as reduction in forage, poor nutrition, and queen failure—are the most probable cause of elevated colony loss rates. Investigators and regulators continue to focus on the possible role that insecticides, particularly the neonicotinoids, may play in honeybee health. Neonicotinoid insecticides are insect neurotoxicants with desirable features such as broad-spectrum activity, low application rates, low mammalian toxicity, upward systemic movement in plants, and versatile application methods. Their distribution throughout the plant, including pollen, nectar, and guttation fluids, poses particular concern for exposure to pollinators. The authors describe how neonicotinoids interact with the nervous system of honeybees and affect individual honeybees in laboratory situations. Because honeybees are social insects, colony effects in semifield and field studies are discussed. The authors conclude with a review of current and proposed guidance in the United States and Europe for assessing the risks of pesticides to honeybees.

Deep Sequencing of Pyrethroid-Resistant Bed Bugs Reveals Multiple Mechanisms of Resistance within a Single Population

A frightening resurgence of bed bug infestations has occurred over the last 10 years in the U.S. and current chemical methods have been inadequate for controlling this pest due to widespread insecticide resistance. Little is known about the mechanisms of resistance present in U.S. bed bug populations, making it extremely difficult to develop intelligent strategies for their control. We have identified bed bugs collected in Richmond, VA which exhibit both kdr-type (L925I) and metabolic resistance to pyrethroid insecticides. Using LD50 bioassays, we determined that resistance ratios for Richmond strain bed bugs were ∼5200-fold to the insecticide deltamethrin. To identify metabolic genes potentially involved in the detoxification of pyrethroids, we performed deep-sequencing of the adult bed bug transcriptome, obtaining more than 2.5 million reads on the 454 titanium platform. Following assembly, analysis of newly identified gene transcripts in both Harlan (susceptible) and Richmond (resistant) bed bugs revealed several candidate cytochrome P450 and carboxylesterase genes which were significantly over-expressed in the resistant strain, consistent with the idea of increased metabolic resistance. These data will accelerate efforts to understand the biochemical basis for insecticide resistance in bed bugs, and provide molecular markers to assist in the surveillance of metabolic resistance.

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.

High Levels of Resistance in the Common Bed Bug, Cimex lectularius (Hemiptera: Cimicidae), to Neonicotinoid Insecticides

Abstract The rapid increase of bed bug populations resistant to pyrethroids demands the development of novel control tactics. Products combining pyrethroids and neonicotinoids have become very popular for bed bug control in the United States, but there are concerns about evolution of resistance to these compounds. Laboratory assays were used to measure the toxicity of topical applications of four neonicotinoids to a susceptible population and three pyrethroid-resistant populations. Activity of esterases, glutathione S-transferases, and cytochrome P450s of all strains was also evaluated. High levels of resistance to four neonicotinoids, acetamiprid, imidacloprid, dinotefuran, and thiamethoxam, relative to the susceptible Fort Dix population, were detected in populations collected from human dwellings in Cincinnati and Michigan. Because activity of detoxifying enzymes was increased in these two populations, our results suggest that these enzymes have some involvement in neonicotinoid resistance, but other resistance mechanisms might be involved as well. Detection of high levels of resistance to neonicotinoids further limits the options for chemical control of bed bugs.

Re-engineering aryl methylcarbamates to confer high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

To identify potential human-safe insecticides against the malaria mosquito we undertook an investigation of the structure-activity relationship of aryl methylcarbamates inhibitors of acetylcholinesterase (AChE). Compounds bearing a β-branched 2-alkoxy or 2-thioalkyl group were found to possess good selectivity for inhibition of Anopheles gambiae AChE over human AChE; up to 530-fold selectivity was achieved with carbamate 11d. A 3D QSAR model is presented that is reasonably consistent with log inhibition selectivity of 34 carbamates. Toxicity of these compounds to live Anopheles gambiae was demonstrated using both tarsal contact (filter paper) and topical application protocols.

Development and comparison of two multi-residue methods for the analysis of select pesticides in honey bees, pollen, and wax by gas chromatography–quadrupole mass spectrometry

One of the hypotheses that may help explain the loss of honey bee colonies worldwide is the increasing potential for exposure of honey bees to complex mixtures of pesticides. To better understand this phenomenon, two multi-residue methods based on different extraction and cleanup procedures have been developed, and compared for the determination of 11 relevant pesticides in honey bees, pollen, and wax by gas chromatography-quadrupole mass spectrometry. Sample preparatory methods included solvent extraction followed by gel permeation chromatography (GPC) cleanup and cleanup using a dispersive solid-phase extraction with zirconium-based sorbents (Z-Sep). Matrix effects, method detection limits, recoveries, and reproducibility were evaluated and compared. Method detection limits (MDL) of the pesticides for the GPC method in honey bees, pollen, and wax ranged from 0.65 to 5.92 ng/g dw, 0.56 to 6.61 ng/g dw, and 0.40 to 8.30 ng/g dw, respectively, while MDLs for the Z-Sep method were from 0.33 to 4.47 ng/g dw, 0.42 to 5.37 ng/g dw, and 0.51 to 5.34 ng/g dw, respectively. The mean recoveries in all matrices and at three spiking concentrations ranged from 64.4% to 149.5% and 71.9% to 126.2% for the GPC and Z-Sep methods, with relative standard deviation between 1.5-25.3% and 1.3-15.9%, respectively. The results showed that the Z-Sep method was more suitable for the determination of the target pesticides, especially chlorothalonil, in bee hive samples. The Z-Sep method was then validated using a series of field-collected bee hive samples taken from honey bee colonies in Virginia.

Honey Bee Gut Microbiome Is Altered by In-Hive Pesticide Exposures

Honey bees (Apis mellifera) are the primary pollinators of major horticultural crops. Over the last few decades, a substantial decline in honey bees and their colonies have been reported. While a plethora of factors could contribute to the putative decline, pathogens, and pesticides are common concerns that draw attention. In addition to potential direct effects on honey bees, indirect pesticide effects could include alteration of essential gut microbial communities and symbionts that are important to honey bee health (e.g., immune system). The primary objective of this study was to determine the microbiome associated with honey bees exposed to commonly used in-hive pesticides: coumaphos, tau-fluvalinate, and chlorothalonil. Treatments were replicated at three independent locations near Blacksburg Virginia, and included a no-pesticide amended control at each location. The microbiome was characterized through pyrosequencing of V2–V3 regions of the bacterial 16S rRNA gene and fungal ITS region. Pesticide exposure significantly affected the structure of bacterial but not fungal communities. The bee bacteriome, similar to other studies, was dominated by sequences derived from Bacilli, Actinobacteria, α-, β-, γ-proteobacteria. The fungal community sequences were dominated by Ascomycetes and Basidiomycetes. The Multi-response permutation procedures (MRPP) and subsequent Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that chlorothalonil caused significant change to the structure and functional potential of the honey bee gut bacterial community relative to control. Putative genes for oxidative phosphorylation, for example, increased while sugar metabolism and peptidase potential declined in the microbiome of chlorothalonil exposed bees. The results of this field-based study suggest the potential for pesticide induced changes to the honey bee gut microbiome that warrant further investigation.

Select Small Core Structure Carbamates Exhibit High Contact Toxicity to ''Carbamate-Resistant'' Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.

Baseline toxicity and field efficacy of metaflumizone on colorado potato beetle (Coleoptera: Chrysomelidae)

ABSTRACT Baseline toxicity levels to a novel semicarbazone insecticide, metaflumizone were established for 25 field populations of Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), from North America. Excluding the susceptible laboratory strain, 50% lethal concentrations of metaflumizone ranged from 0.57 to 1.31 ppm, while response slopes ranged from 1.92 to 4.24 (average = 2.93), and were unrelated to the 50% lethal concentration (r = 0.06; P = 0.76). Beetle populations with known resistance to the neonicotinoid imidacloprid also exhibited the highest LC50 levels to metaflumizone suggesting at least the possibility of cross-resistance. Additional experiments using a potato leaf-dip bioassay as well as field efficacy evaluations confirmed the high level of toxicity of metaflumizone to L. decemlineata and demonstrated a potential benefit of tank mixing a low rate of the pyrethroid esfenvalerate with metaflumizone at one-tenth the recommended field rate. These research findings confirm that metaflumizone is highly active against L. decemlineata larvae and adults and could provide an effective alternative insecticide for potato pest management.

ATP-sensitive inwardly rectifying potassium channel regulation of viral infections in honey bees

Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. In the work described here, we have demonstrated the use of flock house virus (FHV) as a model system for virus infection in bees and revealed an important role for the regulation of the bee antiviral immune response by ATP-sensitive inwardly rectifying potassium (KATP) channels. We have shown that treatment with the KATP channel agonist pinacidil increases survival of bees while decreasing viral replication following infection with FHV, whereas treatment with the KATP channel antagonist tolbutamide decreases survival and increases viral replication. Our results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees.