Heterocyclic Amine-Induced Feeding Deterrence and Antennal Response of Honey Bees.

The productivity and survival of managed honey bee colonies is negatively impacted by a diverse array of interacting factors, including exposure to agrochemicals, such as pesticides. This study investigated the use of volatile heterocyclic amine (HCA) compounds as potential short-term repellents that could be employed as feeding deterrents to reduce the exposure of bees to pesticide-treated plants. Parent and substituted HCAs were screened for efficacy relative to the repellent N,N-diethyl-meta-toluamide (DEET) in laboratory and field experiments. Additionally, electroantennogram (EAG) recordings were conducted to determine the level of antennal response in bees. In video-tracking recordings, bees were observed to spend significantly less time with an HCA-treated food source than an untreated source. In a high-tunnel experiment, the HCA piperidine was incorporated in a feeding station and found to significantly reduce bee visitations relative to an untreated feeder. In field experiments, bee visitations were significantly reduced on melon flowers (Cucumis melo L.) and flowering knapweed (Centaurea stoebe L.) that were sprayed with a piperidine solution, relative to untreated plants. In EAG recordings, the HCAs elicited antennal responses that were significantly different from control or vehicle responses. Overall, this study provides evidence that HCAs can deter individual bees from food sources and suggests that this deterrence is the result of antennal olfactory detection. These findings warrant further study into structure-activity relationships that could lead to the development of short-term repellent compounds that are effective deterrents to reduce the contact of bees to pesticide-treated plants.

Terpenoid-Induced Feeding Deterrence and Antennal Response of Honey Bees.

Multiple interacting stressors negatively affect the survival and productivity of managed honey bee colonies. Pesticides remain a primary concern for beekeepers, as even sublethal exposures can reduce bee immunocompetence, impair navigation, and reduce social communication. Pollinator protection focuses on pesticide application guidelines; however, a more active protection strategy is needed. One possible approach is the use of feeding deterrents that can be delivered as an additive during pesticide application. The goal of this study was to validate a laboratory assay designed to rapidly screen compounds for behavioral changes related to feeding or feeding deterrence. The results of this investigation demonstrated that the synthetic Nasonov pheromone and its terpenoid constituents citral, nerol, and geraniol could alter feeding behavior in a laboratory assay. Additionally, electroantennogram assays revealed that these terpenoids elicited some response in the antennae; however, only a synthetic Nasonov pheromone, citral, and geraniol elicited responses that differed significantly from control and vehicle detections.

Age- and sex-related ABC transporter expression in pyrethroid-susceptible and -resistant Aedes aegypti.

Resistance mechanisms to synthetic insecticides often include point mutations and increased expression of genes encoding detoxification enzymes. Since pyrethroids are the main adulticides used against Aedes aegypti, which vectors pathogens such as Zika virus, understanding resistance to this insecticide class is of significant relevance. We focused on adenosine triphosphate (ATP)-binding cassette (ABC) transporters in the pyrethroid-resistant Puerto Rico (PR) strain of Ae. aegypti. We investigated the expression patterns of six ABC transporters previously characterized as differentially expressed in insecticide-challenged mosquitoes, or increased mRNA expression in pyrethroid-resistant Ae. aegypti, by comparing PR to the Rockefeller (Rock) susceptible strain. No constitutive differential expression between strains was detected, but expression differences for these genes was influenced by sex and age, suggesting that their role is independent from resistance in PR. Instead, ABC transporters may be induced after insecticide exposure. Challenging mosquitoes with deltamethrin, with or without ABC transporter modulators, showed that Rock and PR responded differently, but a contribution of ABC transporters to deltamethrin toxicity is suspected. Moreover, the effect of dexamethasone, which enhanced the inhibition of nerve firing by deltamethrin, was observed using a Drosophila central nervous system preparation, showing synergy of these two compounds through the potential inhibition of ABC transporters.

Bacteria-mediated modification of insecticide toxicity in the yellow fever mosquito, Aedes aegypti.

The incidence of mosquito-borne disease poses a significant threat to human and animal health throughout the world, with effective chemical control interventions limited by widespread insecticide resistance. Recent evidence suggests that gut bacteria of mosquitoes, known to be essential in nutritional homeostasis and pathogen defense, may also play a significant role in facilitating insecticide resistance. This study investigated the extent to which bacteria contribute to the general esterase and cytochrome P450 monooxygenase (P450)-mediated detoxification of the insecticides propoxur and naled, as well as the insecticidal activity of these chemistries to the yellow fever mosquito, Aedes aegypti. Experiments conducted using insecticide synergists that reduce general esterase and P450 activity demonstrate a role for both groups of enzymes in the metabolic detoxification of propoxur and naled. Furthermore, reduction of bacteria in mosquito larvae using broad-spectrum antibiotics was found to decrease the metabolic detoxification of propoxur and naled, suggesting that the bacteria themselves may be contributing to the in vivo metabolic detoxification of these insecticides. This was supported by in vitro assays using culturable gut bacteria isolated from mosquito larvae which demonstrated that the bacteria were capable of reducing insecticide toxicity. More work is needed, however, to fully elucidate the contribution of bacteria in Ae. aegypti larvae to the metabolic detoxification of insecticides.

Vapor delivery of plant essential oils alters pyrethroid efficacy and detoxification enzyme activity in mosquitoes.

The use of synthetic insecticides to limit the spread of mosquito-borne disease faces a number of significant challenges, including insecticide resistance, concerns related to the environmental impact of widespread insecticide use, as well as slowed development of new insecticide chemistries. One important alternative to broadcast insecticides is the use of personal protection strategies to limit contact with vector species, including the use of spatial repellents that can employ synthetic pyrethroids or botanical products to effect control. A currently underexplored area of research involves the investigation of botanical products for their potential to serve as insecticide synergists when delivered as a vapor. This study describes the development of an assay that facilitates the screening of essential oils delivered as a vapor for enhancement of deltamethrin efficacy in both pyrethroid-susceptible and -resistant strains of the vector mosquito species Aedes aegypti. Deltamethrin efficacy was significantly increased following exposure to cinnamon (Cinnamomum cassia), tagetes (Tagetes bipinnata), and sage (Salvia officinalis) oils, while efficacy was significantly decreased following exposure to amyris (Amyris balsamifera) oil. These effects appeared to be mediated by changes in cytochrome P450 activity. This work demonstrates that some plant-derived essential oils delivered as a vapor are capable of increasing the efficacy of deltamethrin similar to classical synergists such as piperonyl butoxide, supporting the use of a real world delivery method instead of traditional contact exposure studies.

Chlorothalonil Exposure Alters Virus Susceptibility and Markers of Immunity, Nutrition, and Development in Honey Bees.

Chlorothalonil is a broad spectrum chloronitrile fungicide that has been identified as one of the most common pesticide contaminants found in managed honey bees (Hymenoptera: Apidae: Apis mellifera L.), their food stores, and the hive environment. While not acutely toxic to honey bees, several studies have identified potential sublethal effects, especially in larvae, but comprehensive information regarding the impact of chlorothalonil on adults is lacking. The goal of this study was to investigate the effects of exposure to a field relevant level of chlorothalonil on honey bee antiviral immunity and biochemical markers of general and social immunity, as well as macronutrient markers of nutrition and morphological markers of growth and development. Chlorothalonil exposure was found to have an effect on 1) honey bee resistance and/or tolerance to viral infection by decreasing the survival of bees following a viral challenge, 2) social immunity, by increasing the level of glucose oxidase activity, 3) nutrition, by decreasing levels of total carbohydrate and protein, and 4) development, by decreasing the total body weight, head width, and wing length of adult nurse and forager bees. Although more research is required to better understand how chlorothalonil interacts with bee physiology to increase mortality associated with viral infections, this study clearly illustrates the sublethal effects of chlorothalonil exposure on bee immunity, nutrition, and development.

Association of age, sex, and pyrethroid resistance status on survival and cytochrome P450 gene expression in Aedes aegypti (L.).

Aedes aegypti is a vector of viruses that negatively impact human health. Insecticide resistance complicates mosquito control efforts, but understanding the mechanisms of resistance can help to improve management practices. This study examined different factors that could influence the interpretation of toxicity bioassays and gene expression studies in A. aegypti, including sex and age, in the context of resistance to pyrethroids. Bioassays using a pyrethroid-resistant strain, Puerto Rico (PR), and a pyrethroid-susceptible strain, Rockefeller (Rock), of A. aegypti were conducted with females and males of three age groups to determine differences in mortality induced by deltamethrin. Overall, strain was the only factor with a significant effect on the LD50. Enzyme assays showed that cytochrome P450 monooxygenase activity in PR was constitutively higher than in Rock, and that pretreatment with the cytochrome P450 inhibitor piperonyl butoxide (PBO) followed by a topical application of deltamethrin (LD25) significantly increased mortality in both strains. Evaluation of the expression levels of seven CYP9J genes previously reported to be involved in pyrethroid resistance revealed that CYP9J10, CYP9J19, and CYP9J28 were more highly expressed in PR than in Rock at all ages of females and males, indicating that they may be essential for resistance. The expression of CYP9J24, CYP9J26, CYP9J27, and CYP9J32 was higher in PR males compared to other groups, including PR females. Significant differences in expression between sexes and strains were also observed as a result of age.

In-Hive Acaricides Alter Biochemical and Morphological Indicators of Honey Bee Nutrition, Immunity, and Development.

The honey bee is a widely managed crop pollinator that provides the agricultural industry with the sustainability and economic viability needed to satisfy the food and fiber needs of our society. Excessive exposure to apicultural pesticides is one of many factors that has been implicated in the reduced number of managed bee colonies available for crop pollination services. The goal of this study was to assess the impact of exposure to commonly used, beekeeper-applied apicultural acaricides on established biochemical indicators of bee nutrition and immunity, as well as morphological indicators of growth and development. The results described here demonstrate that exposure to tau-fluvalinate and coumaphos has an impact on 1) macronutrient indicators of bee nutrition by reducing protein and carbohydrate levels, 2) a marker of social immunity, by increasing glucose oxidase activity, and 3) morphological indicators of growth and development, by altering body weight, head width, and wing length. While more work is necessary to fully understand the broader implications of these findings, the results suggest that reduced parasite stress due to chemical interventions may be offset by nutritional and immune stress.

Interactions between pesticides and pathogen susceptibility in honey bees.

There exist a variety of factors that negatively impact the health and survival of managed honey bee colonies, including the spread of parasites and pathogens, loss of habitat, reduced availability or quality of food resources, climate change, poor queen quality, changing cultural and commercial beekeeping practices, as well as exposure to agricultural and apicultural pesticides both in the field and in the hive. These factors are often closely intertwined, and it is unlikely that a single stressor is driving colony losses. There is a growing consensus, however, that increasing prevalence of parasites and pathogens are among the most significant threats to managed bee colonies. Unfortunately, improper management of hives by beekeepers may exacerbate parasite populations and disease transmission. Furthermore, research continues to accumulate that describes the complex and largely harmful interactions that exist between pesticide exposure and bee immunity. This brief review summarizes our progress in understanding the impact of pesticide exposure on bees at the individual, colony, and community level.

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.

Amitraz and its metabolite modulate honey bee cardiac function and tolerance to viral infection.

The health and survival of managed honey bee (Apis mellifera) colonies are affected by multiple factors, one of the most important being the interaction between viral pathogens and infestations of the ectoparasitic mite Varroa destructor. Currently, the only effective strategy available for mitigating the impact of viral infections is the chemical control of mite populations. Unfortunately, the use of in-hive acaricides comes at a price, as they can produce sublethal effects that are difficult to quantify, but may ultimately be as damaging as the mites they are used to treat. The goal of this study was to investigate the physiological and immunological effects of the formamidine acaricide amitraz and its primary metabolite in honey bees. Using flock house virus as a model for viral infection, this study found that exposure to a formamidine acaricide may have a negative impact on the ability of honey bees to tolerate viral infection. Furthermore, this work has demonstrated that amitraz and its metabolite significantly alter honey bee cardiac function, most likely through interaction with octopamine receptors. The results suggest a potential drawback to the in-hive use of amitraz and raise intriguing questions about the relationship between insect cardiac function and disease tolerance.

ATP-sensitive inwardly rectifying potassium channel modulators alter cardiac function in honey bees.

ATP-sensitive inwardly rectifying potassium (KATP) channels couple cellular metabolism to the membrane potential of the cell and play an important role in a variety of tissue types, including the insect dorsal vessel, making them a subject of interest not only for understanding invertebrate physiology, but also as a potential target for novel insecticides. Most of what is known about these ion channels is the result of work performed in mammalian systems, with insect studies being limited to only a few species and physiological systems. The goal of this study was to investigate the role that KATP channels play in regulating cardiac function in a model social insect, the honey bee (Apis mellifera), by examining the effects that modulators of these ion channels have on heart rate. Heart rate decreased in a concentration-dependent manner, relative to controls, with the application of the KATP channel antagonist tolbutamide and KATP channel blockers barium and magnesium, whereas heart rate increased with the application of a low concentration of the KATP channel agonist pinacidil, but decreased at higher concentrations. Furthermore, pretreatment with barium magnified the effects of tolbutamide treatment and eliminated the effects of pinacidil treatment at select concentrations. The data presented here confirm a role for KATP channels in the regulation of honey bee dorsal vessel contractions and provide insight into the underlying physiology that governs the regulation of bee cardiac function.

Dissection and Observation of Honey Bee Dorsal Vessel for Studies of Cardiac Function.

The European honey bee, Apis mellifera L., is a valuable agricultural and commercial resource noted for producing honey and providing crop pollination services, as well as an important model social insect used to study memory and learning, aging, and more. Here we describe a detailed protocol for the dissection of the dorsal abdominal wall of a bee in order to visualize its dorsal vessel, which serves the role of the heart in the insect. A successful dissection will expose a functional heart that, under the proper conditions, can maintain a steady heartbeat for an extended period of time. This allows the investigator to manipulate heart rate through the application of cardiomodulatory compounds to the dorsal vessel. By using either a digital microscope or a microscope equipped with a digital camera, the investigator can make video recordings of the dorsal vessel before and after treatment with test compounds. The videos can then be scored at a time convenient to the user in order to determine changes in heart rate, as well as changes in the pattern of heartbeats, following treatment. The advantages of this protocol are that it is relatively inexpensive to set up, easy to learn, requires little space or equipment, and takes very little time to conduct.

Mosquito-borne viruses and suppressors of invertebrate antiviral RNA silencing.

The natural maintenance cycles of many mosquito-borne viruses require establishment of persistent non-lethal infections in the invertebrate host. While the mechanisms by which this occurs are not well understood, antiviral responses directed by small RNAs are important in modulating the pathogenesis of viral infections in disease vector mosquitoes. In yet another example of an evolutionary arms race between host and pathogen, some plant and insect viruses have evolved to encode suppressors of RNA silencing (VSRs). Whether or not mosquito-borne viral pathogens encode VSRs has been the subject of debate. While at first there would seem to be little evolutionary benefit to mosquito-borne viruses encoding proteins or sequences that strongly interfere with RNA silencing, we present here a model explaining how the expression of VSRs by these viruses in the vector might be compatible with the establishment of persistence. We also discuss the challenges associated with interrogating these viruses for the presence of suppressor proteins or sequences, as well as the candidates that have been identified in the genomes of mosquito-borne pathogens thus far.

Rational design of fatty acid amide hydrolase inhibitors that act by covalently bonding to two active site residues.

The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1-6 h time course monitored.

Inhibition of monoacylglycerol lipase attenuates nonsteroidal anti-inflammatory drug-induced gastric hemorrhages in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type 2 (CB(2)), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(2) and D(2). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1ß, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(2) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.

CB1 receptors mediate rimonabant-induced pruritic responses in mice: investigation of locus of action.

RATIONALE: Cannabinoids have recently been identified as potential neuronal modulators of pruritic response, representing a potential target in the treatment of itch associated with a variety of pathophysiologic conditions. While the selective CB(1) receptor antagonist rimonabant is an established pruritic agent in both animal and clinical testing, its receptor mechanism of action and anatomical loci remain unclear. OBJECTIVE: The purpose of this study was to determine whether CB(1) receptor blockade is critical to rimonabant-induced scratching and to identify differences in scratching response based on different routes of administration. Furthermore, experiments were designed to elucidate any evidence as to whether rimonabant elicits scratching behavior through common immunologic hypersensitivity mechanisms. RESULTS: Rimonabant was equally effective at producing scratching via intraperitoneal and local subcutaneous injection. This compound also produced an intense scratching response when administered intrathecally, but had no effects after intracerebroventricular administration. Repeated administration of rimonabant led to a decreased magnitude of scratching. While rimonabant-induced scratching was not attenuated either by pretreatment with the H(1) receptor antagonist loratadine or in mast cell-deficient mice, it lacked efficacy in CB(1) (-/-) mice. CONCLUSIONS: Rimonabant is a potent and fully effective pruritogen when administered spinally or systemically and requires CB(1) receptors to induce scratching, suggesting an important spinal CB(1) receptor component of action. The lack of responsiveness to H(1) antagonism or mast cell deficiency supports previous findings that cannabinoids modulate itch through neuronal mechanisms, and not by traditional hypersensitivity activation.

Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay.

Cannabinoids have long been shown to have a range of potential therapeutic effects, including antiemetic actions, analgesia, and anxiolysis. However, psychomimetic and memory disruptive side effects, as well as the potential for abuse and dependence, have restricted their clinical development. Endogenous cannabinoids (i.e., endocannabinoids; eCBs), such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are produced throughout the limbic system and other brain regions associated with emotionality and are believed to modulate behavioral responses to stress-related conditions. AEA and 2-AG are rapidly metabolized by the respective enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). Accordingly, inhibition of each enzyme increases brain levels of the appropriate eCB. Although FAAH inhibition has been established to decrease anxiety-like behavior, the role of 2-AG has been difficult to ascertain until the recent synthesis of JZL184, a potent and selective MAGL inhibitor. In the present study, we investigated the effects of inhibiting FAAH or MAGL on anxiety-like behavior in marble burying, a model of repetitive, compulsive behaviors germane to anxiety disorders such as obsessive-compulsive disorder. The FAAH inhibitor PF-3845, the MAGL inhibitor JZL184, and the benzodiazepine diazepam decreased marble burying at doses that did not affect locomotor activity. In contrast, Δ9-tetrahydrocannabinol (THC), the primary psychoactive constituent of marijuana, did not consistently reduce marble burying without also eliciting profound decreases in locomotor behavior. The CB1 cannabinoid receptor antagonist rimonabant blocked the reduction in marble burying caused by FAAH and MAGL inhibitors, but not by diazepam, indicating a CB1 receptor mechanism of action. These data indicate that elevation of AEA or 2-AG reduces marble burying behavior and suggest that their catabolic enzymes represent potential targets for the development of new classes of pharmacotherapeutics to treat anxiety-related disorders.