Potassium ion channels as a molecular target to reduce virus infection and mortality of honey bee colonies.

Declines in managed honey bee populations are multifactorial but closely associated with reduced virus immunocompetence and thus, mechanisms to enhance immune function are likely to reduce viral infection rates and increase colony viability. However, gaps in knowledge regarding physiological mechanisms or 'druggable' target sites to enhance bee immunocompetence has prevented therapeutics development to reduce virus infection. Our data bridge this knowledge gap by identifying ATP-sensitive inward rectifier potassium (KATP) channels as a pharmacologically tractable target for reducing virus-mediated mortality and viral replication in bees, as well as increasing an aspect of colony-level immunity. Bees infected with Israeli acute paralysis virus and provided KATP channel activators had similar mortality rates as uninfected bees. Furthermore, we show that generation of reactive oxygen species (ROS) and regulation of ROS concentrations through pharmacological activation of KATP channels can stimulate antiviral responses, highlighting a functional framework for physiological regulation of the bee immune system. Next, we tested the influence of pharmacological activation of KATP channels on infection of 6 viruses at the colony level in the field. Data strongly support that KATP channels are a field-relevant target site as colonies treated with pinacidil, a KATP channel activator, had reduced titers of seven bee-relevant viruses by up to 75-fold and reduced them to levels comparable to non-inoculated colonies. Together, these data indicate a functional linkage between KATP channels, ROS, and antiviral defense mechanisms in bees and define a toxicologically relevant pathway that can be used for novel therapeutics development to enhance bee health and colony sustainability in the field.

Bioinsecticidal activity of cajeput oil to pyrethroid-susceptible and -resistant mosquitoes.

Mosquito-borne diseases are a significant threat to human health. The frequent and repetitive application of insecticides can result in the selection of resistant mosquito populations leading to product failures for reducing community disease transmission. It is important that new interventions are discovered and developed for reducing mosquito populations and, in turn, protecting human health. Plant essential oils are promising chemical interventions for reducing mosquito populations. The myrtle family, Myrtaceae, has numerous species to be studied as potential bioinsecticides. Here, we combined toxicological, biochemical, and neurophysiological approaches to provide evidence for cajeput oil and terpene constituents to elicit bioinsecticidal activity to pyrethroid-susceptible and -resistant Aedes aegypti. We show cajeput oil terpenes to enhance cAMP production, increase ACh levels, inhibit in vivo and in vitro AChE activity, and disrupt spike discharge frequencies of the mosquito CNS. This study presents the first report on the bioinsecticidal activity of cajeput oil terpenes to pyrethroid-susceptible and -resistant mosquitoes and provides comparative data for the octopaminergic system as a putative molecular target for the bioinsecticides with implications for resistance management.

Acute toxicity of atrazine, alachlor, and chlorpyrifos mixtures to honey bees.

The acute toxicity of chlorpyrifos and chlorpyrifos-oxon (organophosphorothioate insecticides) was examined alone and in combination with atrazine (triazine herbicide) and alachlor (chloroacetanilide herbicide) to honey bees (Apis mellifera). Atrazine and alachlor were observed to not be acutely toxic to bees at doses up to 10 and 4 µg per bee, respectively. However, atrazine significantly increased chlorpyrifos toxicity by 3-fold while reducing chlorpyrifos-oxon toxicity by 1.8-fold. These changes in toxicity are correlated with significant 1.3- and 1.2-fold inhibition of acetylcholinesterase (AChE) activity in bees exposed to chlorpyrifos and chlorpyrifos-oxon, respectively. Atrazine significantly increased cytochrome P450, general esterase, and glutathione S-transferase (GST) activities by 1.5-, 1.2-, and 1.2- fold respectively, in bees compared to untreated individuals. Alachlor increased chlorpyrifos toxicity by 2.5-fold but did not affect the toxicity of chlorpyrifos-oxon. Exposure to alachlor and chlorpyrifos did not affect AChE compared to chlorpyrifos alone. However, exposure to chlorpyrifos-oxon and alachlor significantly increased acetylcholinesterase (AChE) activity by 1.4-fold. GST activity, but not P450 or general esterases, was significantly increased in bees exposed to alachlor. These data provide evidence that triazine and chloroacetanilide herbicide exposure alters detoxification enzyme activity and, in turn, alters the sensitivity of bees to organophosphorothioate insecticides. Importantly, these data can be used to guide future studies aiming to test safety profiles for pollinators and expand regulatory framework required for pesticide registration.

Bee-safe peptidomimetic acaricides achieved by comparative genomics.

The devastating Varroa mite (Varroa destructor Anderson and Trueman) is an obligatory ectoparasite of the honey bee, contributing to significant colony losses in North America and throughout the world. The limited number of conventional acaricides to reduce Varroa mites and prevent disease in honey bee colonies is challenged with wide-spread resistance and low target-site selectivity. Here, we propose a biorational approach using comparative genomics for the development of honey bee-safe and selective acaricides targeting the Varroa mite-specific neuropeptidergic system regulated by proctolin, which is lacking in the honey bee. Proctolin is a highly conserved pentapeptide RYLPT (Arg-Tyr-Leu-Pro-Thr) known to act through a G protein-coupled receptor to elicit myotropic activity in arthropod species. A total of 33 different peptidomimetic and peptide variants were tested on the Varroa mite proctolin receptor. Ligand docking model and mutagenesis studies revealed the importance of the core aromatic residue Tyr2 in the proctolin ligand. Peptidomimetics were observed to have significant oral toxicity leading to the paralysis and death of Varroa mites, while there were no negative effects observed for honey bees. We have demonstrated that a taxon-specific physiological target identified by advanced genomics information offers an opportunity to develop Varroa mite-selective acaricides, hence, expedited translational processes.

Pyrethroid insecticide and milkweed cardenolide interactions on detoxification enzyme activity and expression in monarch caterpillars.

Declines of the monarch butterfly population have prompted large-scale plantings of milkweed to restore the population. In North America, there are >73 species of milkweed to choose from for these nationwide plantings. However, it is unclear how different milkweed species affect monarch caterpillar physiology, particularly detoxification enzyme activity and gene expression, given the highly variable cardenolide composition across milkweed species. Here, we investigate the effects of a high cardenolide, tropical milkweed species and a low cardenolide, swamp milkweed species on pyrethroid sensitivity as well as detoxification enzyme activity and expression in monarch caterpillars. Caterpillars fed on each species through the fifth-instar stage and were topically treated with bifenthrin after reaching this final-instar stage. Esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were quantified as well as the expression of selected esterase, glutathione S-transferase, ABC transporter, and cytochrome P450 monooxygenase transcripts. There were no significant differences in survival 24 h after treatment with bifenthrin. However, bifenthrin significantly increased glutathione S-transferase activity in caterpillars feeding on tropical milkweed and significantly decreased esterase activity in caterpillars feeding on tropical and swamp milkweed. Significant differential expression of ABC transporter, glutathione S-transferase, and esterase genes was observed for caterpillars feeding on tropical and swamp milkweed and not receiving bifenthrin treatment. Furthermore, significant differential expression of glutathione S-transferase and esterase genes was observed for bifenthrin-treated and -untreated caterpillars feeding on tropical milkweed relative to swamp milkweed. These results suggest that feeding on different milkweed species can affect detoxification and development mechanisms with which monarch caterpillars rely on to cope with their environment.

Testing new compounds for efficacy against Varroa destructor and safety to honey bees (Apis mellifera).

BACKGROUND: Varroa destructor is among the greatest threats to honey bee health worldwide. Acaricides used to control Varroa are becoming increasingly ineffective due to resistance issues, prompting the need for new compounds that can be used for control purposes. Ideally, such compounds would exhibit high toxicity to Varroa while maintaining relatively low toxicity to bees and beekeepers. We characterized the lethal concentrations (LC50 ) of amitraz, matrine, FlyNap®, the experimental carbamates 2-((2-ethylbutyl)thio)phenyl methylcarbamate (1) and 2-(2-ethylbutoxy)phenyl methylcarbamate (2), and dimethoate (positive control) for Varroa using a glass vial assay. The test compounds also were applied to honey bees using an acute contact toxicity assay to determine the adult bee LD50 for each compound. RESULTS: Amitraz was the most toxic compound to Varroa, but carbamate 2 was nearly as active (within 2-fold) and the most selective due to its lower bee toxicity, demonstrating its promise as a Varroa control. While carbamate 1 was less toxic to honey bees than was amitraz, it was also 4.7-fold less toxic to the mites. Both matrine and FlyNap® were relatively ineffective at killing Varroa and were moderately toxic to honey bees. CONCLUSION: Additional testing is required to determine if carbamate 2 can be used as an effective Varroa control. As new chemical treatments are identified, it will be necessary to determine how they can be utilized best alongside other control techniques as part of an integrated pest management program. © 2021 Society of Chemical Industry.

Toxicological analysis of stilbenes against the fall armyworm, Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a global pest of multiple economically important row crops and the development of resistance to commercially available insecticidal classes has inhibited FAW control. Thus, there is a need to identify chemical scaffolds that can provide inspiration for the development of novel insecticides for FAW management. This study aimed to assess the sensitivity of central neurons and susceptibility of FAW to chloride channel modulators to establish a platform for repurposing existing insecticides or designing new chemicals capable of controlling FAW. Potency of select chloride channel modulators were initially studied against FAW central neuron firing rate and rank order of potency was determined to be fipronil > lindane > Z-stilbene > DIDS > GABA > E-stilbene. Toxicity bioassays identified fipronil and lindane as the two most toxic modulators studied with topical LD50's of 41 and 75 ng/mg of caterpillar, respectively. Interestingly, Z-stilbene was toxic at 300 ng/mg of caterpillar, but no toxicity was observed with DIDS or E-stilbene. The significant shift in potency between stilbene isomers indicates structure-activity relationships between stilbene chemistry and the binding site in FAW may exist. The data presented in this study defines the potency of select chloride channel modulators to FAW neural activity and survivorship to establish a platform for development of novel chemical agents to control FAW populations. Although stilbenes may hold promise for insecticide development, the low toxicity of the scaffolds tested in this study dampen enthusiasm for their development into FAW specific insecticides.

Cardenolide, Potassium, and Pyrethroid Insecticide Combinations Reduce Growth and Survival of Monarch Butterfly Caterpillars (Lepidoptera: Nymphalidae).

The monarch butterfly, Danaus plexippus L., has evolved to be insensitive to milkweed cardenolides via genetic modifications of Na+/K+-ATPase. There is concern for insecticide exposures near agriculture, with little information on monarch caterpillar toxicology. It is unclear how cardenolide insensitivity may affect the sensitivity of monarch caterpillars to pyrethroid insecticides. Additionally, potassium fertilizers may affect monarch caterpillar physiology and cardenolide sequestration. Here, we investigated the growth, survival, and development of caterpillars exposed to the cardenolide ouabain, bifenthrin, and potassium chloride (KCl) alone and in combination. Caterpillars were either exposed to 1) ouabain from third- to fifth-instar stage, 2) KCl at fifth-instar stage, 3) KCl and bifenthrin at fifth-instar stage, or 4) combinations of ouabain at third-instar stage + KCl + bifenthrin at fifth-instar stage. Caterpillar weight, diet consumption, frass, and survival were recorded for the duration of the experiments. It was observed that 1-3 mg ouabain/g diet increased body weight and diet consumption, whereas 50 mg KCl/g diet decreased body weight and diet consumption. Caterpillars feeding on KCl and treated with 0.2 µg/µl bifenthrin consumed significantly less diet compared to individuals provided untreated diet. However, there was no effect on survival or body weight. Combinations of KCl + ouabain did not significantly affect caterpillar survival or body weight following treatment with 0.1 µg/µl bifenthrin. At the concentrations tested, there were no effects observed for bifenthrin sensitivity with increasing cardenolide or KCl concentrations. Further studies are warranted to understand how milkweed-specific cardenolides, at increasing concentrations, and agrochemical inputs can affect monarch caterpillar physiology near agricultural landscapes.

Pyrethroid Exposure Reduces Growth and Development of Monarch Butterfly (Lepidoptera: Nymphalidae) Caterpillars.

Insecticide exposure has been identified as a contributing stressor to the decline in the North American monarch butterfly Danaus plexippus L. (Lepidoptera: Nymphalidae) population. Monarch toxicity data are currently limited and available data focuses on lethal endpoints. This study examined the 72-h toxicity of two pyrethroid insecticides, bifenthrin and ß-cyfluthrin, and their effects on growth and diet consumption. The toxicity of bifenthrin to caterpillars was lower than ß-cyfluthrin after 72 h. Survival was the most sensitive endpoint for bifenthrin, but diet consumption and caterpillar growth were significantly reduced at sublethal levels of ß-cyfluthrin. Using AgDRIFT spray drift assessment, the aerial application of bifenthrin or ß-cyfluthrin is predicted to pose the greatest risk to fifth-instar caterpillars, with lethal insecticide deposition up to 28 m for bifenthrin and up to 23 m for ß-cyfluthrin from treated edges of fields. Low boom ground applications are predicted to reduce distances of lethal insecticide exposure to 2 m from the treated field edge for bifenthrin and ß-cyfluthrin. Growth and survival of fifth-instar monarch caterpillars developing within the margins of a treated field may be significantly impacted following foliar applications of bifenthrin or ß-cyfluthrin. These findings provide evidence that pyrethroid insecticides commonly used for soybean pest control are a potential risk to monarch caterpillars in agricultural landscapes.

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.

Reduced neuronal sensitivity and susceptibility of the fall armyworm, Spodoptera frugiperda, to pyrethroids in the absence of known knockdown mutations.

Neurophysiological recordings were employed to quantify neuronal sensitivity to neurotoxic insecticides and assessed toxicity across field and laboratory fall armyworm (FAW) populations. Topical toxicity resistance ratios (RR) in field-collected FAW was 767-fold compared to laboratory strains and, importantly, a 1750-fold reduction in potency was observed for λ-cyhalothrin in neurophysiological assays. Field collected FAW were found to have a RR of 12 to chlorpyrifos when compared to the susceptible strain and was 8-fold less sensitive in neurophysiological assays. Surprisingly, there were no point mutations identified in the voltage-gated sodium channel known to cause pyrethroid resistance. For acetylcholinesterase, FAW had more than 80% of their nucleotide sequences consistent with A201 and F290 of the susceptible strains although 60% of the tested population was heterozygous for the G227A mutation. These data indicate that point mutations did not contribute to the high level of pyrethroid resistance and nerve insensitivity in this population of field collected FAW. Additionally, these data suggest the kdr phenotype only explains a portion of the heritable variation in FAW resistance and indicates kdr is not the only predictor of high pyrethroid resistance. Phenotypic assays, such as toxicity bioassays or neurophysiological recordings, using field-collected populations are necessary to reliably predict resistant phenotypes and product failures.

Voltage-gated chloride channel blocker DIDS as an acaricide for Varroa mites.

The Varroa mite is a primary driver behind periodical losses of honey bee colonies. These mites require honey bees for food and reproduction and, in turn, elicit physiological deficiencies and diseases that compromise colony health. Current acaricides for Varroa mite control, such as Apistan® (the pyrethroid tau-fluvalinate), CheckMite+® (the organophosphate coumaphos), and Apivar® (the formamidine amitraz) target the nervous system, can have adverse health effects on honey bees, and have limited effectiveness due to reported resistance issues. New target sites are needed to circumvent these obstacles in Varroa mite management, and voltage-gated chloride channels (VGCCs) are promising candidates due to their important role in the maintenance of nerve and muscle excitability in arthropod pests. Toxicological analysis of Varroa mites sensitive to tau-fluvalinate and coumaphos and Varroa mites with reduced sensitivity to these acaricides showed a significant increase in metabolic detoxification enzyme activities for the latter. Acetylcholinesterase activity in the Varroa mites exhibiting reduced mortality to coumaphos was significantly less sensitive to coumaphos-oxon compared to coumaphos-sensitive Varroa mites, which suggests target-site insensitivity to the acaricide. Voltage-gated chloride channel blocker DIDS had significantly greater field efficacy compared to Apistan® and CheckMite+® against Varroa mites from honey bee hives where tau-fluvalinate and coumaphos were observed to be ineffective, respectively. These data suggest that DIDS, and potentially other stilbene chemistries, might serve as candidates for continued field efficacy testing of alternative acaricides in apiaries where Apistan®- and CheckMite+® efficacy has been. reduced or lost for Varroa mites.

Comparative effects of technical-grade and formulated chlorantraniliprole to the survivorship and locomotor activity of the honey bee, Apis mellifera (L.).

BACKGROUND: The loss of honey bee colonies is a nationally recognized problem that demands attention from both the scientific community and the beekeeping industry. One outstanding threat is the unintended exposure of these pollinators to agricultural pesticides. Anthranilic diamides, such as chlorantraniliprole, are registered for use in stone and pome fruits, vegetables, turf, and grains. There are few publicly available studies that provide an analysis of chlorantraniliprole effects on the survivorship and locomotion activity of beneficial, pollinating insects such as honey bees. The data gathered in this study provide the acute toxicity, 30-day survivorship, and locomotor activity of honey bees exposed to technical-grade chlorantraniliprole and three formulated products with chlorantraniliprole as the active ingredient. RESULTS: Neither the technical-grade nor the formulated products of chlorantraniliprole were acutely toxic to honey bees following 4 or 72h treatments at the tested concentrations. A 4 h treatment of technical-grade and formulated chlorantraniliprole did not significantly affect the 30-day survivorship, although significantly higher mortality was observed after 30 days for bees receiving a 72 h treatment of technical-grade chlorantraniliprole and two formulated products. The locomotion activity, or total walking distance, of bees receiving a 4 h treatment of one chlorantraniliprole formulation was significantly reduced, with these individuals recovering their normal locomotion activity at 48 h post exposure. Conversely, there was observed lethargic behavior and significantly reduced walking distances for bees provided with a 72 h treatment of technical-grade chlorantraniliprole and each formulated product. CONCLUSION: This study provides evidence for the effect of long-term exposure of chlorantraniliprole on the survivorship and locomotor activity of honey bees. Bees receiving a more field-relevant short-term exposure survived and moved similarly to untreated bees, reiterating the relative safety of chlorantraniliprole exposure to adult honey bees at recommended label concentrations. © 2020 Society of Chemical Industry.

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.

Sublethal Effects of the Insecticide Pyrifluquinazon on the European Honey Bee (Hymenoptera: Apidae).

Pyrifluquinazon (PQZ) is an Insecticide Resistance Action Committee (IRAC) Group 9 insecticide that has recently been registered for use in the United States for control of soft-bodied sucking insect pests. Although it has been classified as practically nontoxic to honey bees, Apis mellifera L. (Hymenoptera: Apidae), based on acute contact bioassays, additional information on sublethal effects of this insecticide on honey bees is lacking. Using a combination of laboratory assays with video movement tracking software and near-field evaluations of colonies foraging in a high-tunnel experiment, we determined that, when fed PQZ at a concentration of 84 mg active ingredient (ai)/liter (= ppm) in sugar water, a reduction in overall movement by the foraging worker bees was observed. However, when provided with honey reserves in the hive, honey bees rejected the PQZ-treated sugar water. These results indicate that, if ingested at levels of 84 mg ai/liter, PQZ could have a negative effect on honey bee behavior; however, honey bee workers appear to be able to detect the presence of PQZ in their food and reject it.