Christopher A. Mullin

Colony Collapse Disorder: A Descriptive Study

Background Over the last two winters, there have been large-scale, unexplained losses of managed honey bee (Apis mellifera L.) colonies in the United States. In the absence of a known cause, this syndrome was named Colony Collapse Disorder (CCD) because the main trait was a rapid loss of adult worker bees. We initiated a descriptive epizootiological study in order to better characterize CCD and compare risk factor exposure between populations afflicted by and not afflicted by CCD. Methods and Principal Findings Of 61 quantified variables (including adult bee physiology, pathogen loads, and pesticide levels), no single measure emerged as a most-likely cause of CCD. Bees in CCD colonies had higher pathogen loads and were co-infected with a greater number of pathogens than control populations, suggesting either an increased exposure to pathogens or a reduced resistance of bees toward pathogens. Levels of the synthetic acaricide coumaphos (used by beekeepers to control the parasitic mite Varroa destructor) were higher in control colonies than CCD-affected colonies. Conclusions/Significance This is the first comprehensive survey of CCD-affected bee populations that suggests CCD involves an interaction between pathogens and other stress factors. We present evidence that this condition is contagious or the result of exposure to a common risk factor. Potentially important areas for future hypothesis-driven research, including the possible legacy effect of mite parasitism and the role of honey bee resistance to pesticides, are highlighted.

High levels of miticides and agrochemicals in North American apiaries: implications for honey bee health.

Background Recent declines in honey bees for crop pollination threaten fruit, nut, vegetable and seed production in the United States. A broad survey of pesticide residues was conducted on samples from migratory and other beekeepers across 23 states, one Canadian province and several agricultural cropping systems during the 2007–08 growing seasons. Methodology/Principal Findings We have used LC/MS-MS and GC/MS to analyze bees and hive matrices for pesticide residues utilizing a modified QuEChERS method. We have found 121 different pesticides and metabolites within 887 wax, pollen, bee and associated hive samples. Almost 60% of the 259 wax and 350 pollen samples contained at least one systemic pesticide, and over 47% had both in-hive acaricides fluvalinate and coumaphos, and chlorothalonil, a widely-used fungicide. In bee pollen were found chlorothalonil at levels up to 99 ppm and the insecticides aldicarb, carbaryl, chlorpyrifos and imidacloprid, fungicides boscalid, captan and myclobutanil, and herbicide pendimethalin at 1 ppm levels. Almost all comb and foundation wax samples (98%) were contaminated with up to 204 and 94 ppm, respectively, of fluvalinate and coumaphos, and lower amounts of amitraz degradates and chlorothalonil, with an average of 6 pesticide detections per sample and a high of 39. There were fewer pesticides found in adults and brood except for those linked with bee kills by permethrin (20 ppm) and fipronil (3.1 ppm). Conclusions/Significance The 98 pesticides and metabolites detected in mixtures up to 214 ppm in bee pollen alone represents a remarkably high level for toxicants in the brood and adult food of this primary pollinator. This represents over half of the maximum individual pesticide incidences ever reported for apiaries. While exposure to many of these neurotoxicants elicits acute and sublethal reductions in honey bee fitness, the effects of these materials in combinations and their direct association with CCD or declining bee health remains to be determined.

Pesticides and honey bee toxicity - USA*

Until 1985 discussions of pesticides and honey bee toxicity in the USA were focused on pesticides applied to crops and the unintentional exposure of foraging bees to them. The recent introduction of arthropod pests of honey bees, Acarapis woodi (1984), Varroa destructor (1987), and Aethina tumida (1997), to the USA have resulted in the intentional introduction of pesticides into beehives to suppress these pests. Both the unintentional and the intentional exposure of honey bees to pesticides have resulted in residues in hive products, especially beeswax. This review examines pesticides applied to crops, pesticides used in apiculture and pesticide residues in hive products. We discuss the role that pesticides and their residues in hive products may play in colony collapse disorder and other colony problems. Although no single pesticide has been shown to cause colony collapse disorder, the additive and synergistic effects of multiple pesticide exposures may contribute to declining honey bee health.ZusammenfassungNeuere systemisch wirkende Pestizide, einschließlich der Neonikotinoide (z. B. Imidacloprid) und Phenylpyrazole (z. B. Fipronil) finden in den USA verbreitete Anwendung im Pflanzenschutz. Das Gefährdungspotenzial von Bienen durch diese Präparate unterscheidet sich von dem traditioneller Pestizidanwendungen, bei denen die hauptsächliche Sorge der akuten Giftigkeit galt. Im Hinblick auf die Verordnungen zu Pestiziden in den USA wurden die Folgen von chronischer und sublethaler Belastung durch systemische Mittel bisher nicht umfassend in Betracht gezogen, obwohl die Sachlage, was diese Präparate betrifft, gegenwärtig von der Umweltbehörde (EPA) begutachtet wird. Zahlreiche in den USA angebaute Pflanzen wurden genetisch verändert, um entweder insektizid wirkende Bt Toxine oder Herbizidresistenz zu exprimieren. Insektizid wirkende Bt Toxine scheinen jedoch spezifisch toxisch für Ertragsschädlinge zu sein und können daher den Bienen nützen, indem sie die Anwendung traditioneller Pestizide reduzieren.Bis zur Einführung von arthropoden Bienenschädlingen in die USA in der Mitte der achtziger Jahre wurden Bienen den verschiedenen Pestiziden nur unbeabsichtigt ausgesetzt, während sie auf gespritzten Pflanzen sammelten. Die Notwendigkeit, Bienenschädlinge, besonders die Varroamilbe (Varroa destructor), zu bekämpfen, erfordert seitdem jedoch oft eine absichtliche Anwendung von Pestiziden in Bienenvölkern. Tau-Fluvalinat und Coumaphos, jeweils in Streifenform angewendet, sind in den USA immer noch für die Anwendung in Bienenvölkern zugelassen, obwohl die Wirksamkeit dieser Substanzen gegen Varroamilben durch die Entwicklung von Resistenzen vermindert wurde. Ein neues Varroazid, Fenpyroximate, wurde in einigen Staaten zur Anwendung zugelassen. Essentielle Öle, einschließlich Thymol und Menthol, sind ebenso wie Ameisensäure zur Anwendung in der Verdampfung zugelassen. Oxalsäure ist nur in Kanada, jedoch nicht in den USA zugelassen.Über 150 verschiedene Pestizide wurden in Proben aus Bienenständen in den USA gefunden. Von Imkern eingesetzte Pestizide werden tendenziell öfter im Wachs der Völker nachgewiesen, von wo aus Pollen, Bienenbrot und Honig damit kontaminiert werden. Auf der anderen Seite werden Pestizide, vor allem Fungizide, die nicht in Bienenvölkern eingesetzt werden, tendenziell am häufigsten in Pollen nachgewiesen und kontaminieren das Wachs nur dann, wenn sie eingelagert werden. Da Honigbienen den sublethalen Konzentrationen zahlreicher Pestizide gleichzeitig ausgesetzt sind, wird zusätzliche Forschung zur Aufklärung synergistischer Effekte bei chronischer sublethaler Belastung mit mehreren Pesitziden benötigt.

Four common pesticides, their mixtures and a formulation solvent in the hive environment have high oral toxicity to honey bee larvae.

Recently, the widespread distribution of pesticides detected in the hive has raised serious concerns about pesticide exposure on honey bee (Apis mellifera L.) health. A larval rearing method was adapted to assess the chronic oral toxicity to honey bee larvae of the four most common pesticides detected in pollen and wax - fluvalinate, coumaphos, chlorothalonil, and chloropyrifos - tested alone and in all combinations. All pesticides at hive-residue levels triggered a significant increase in larval mortality compared to untreated larvae by over two fold, with a strong increase after 3 days of exposure. Among these four pesticides, honey bee larvae were most sensitive to chlorothalonil compared to adults. Synergistic toxicity was observed in the binary mixture of chlorothalonil with fluvalinate at the concentrations of 34 mg/L and 3 mg/L, respectively; whereas, when diluted by 10 fold, the interaction switched to antagonism. Chlorothalonil at 34 mg/L was also found to synergize the miticide coumaphos at 8 mg/L. The addition of coumaphos significantly reduced the toxicity of the fluvalinate and chlorothalonil mixture, the only significant non-additive effect in all tested ternary mixtures. We also tested the common ‘inert’ ingredient N-methyl-2-pyrrolidone at seven concentrations, and documented its high toxicity to larval bees. We have shown that chronic dietary exposure to a fungicide, pesticide mixtures, and a formulation solvent have the potential to impact honey bee populations, and warrants further investigation. We suggest that pesticide mixtures in pollen be evaluated by adding their toxicities together, until complete data on interactions can be accumulated.

Chalcone oxides—potent selective inhibitors of cytosolic epoxide hydrolase☆

The systematic screening of over 150 compounds for inhibitory activity on mammalian cytosolic epoxide hydrolase led to identification of chalcone oxide (trans-1-benzoyl-2-phenyloxirane) as an optimal inhibitory structure. Important structural features for inhibition include two hydrophobic moieties preferably orientating in a trans manner from an electrophilic center such as an activated olefin or epoxide, with the epoxide giving maximal activity. Synthesis of chalcone oxide derivatives bearing a single p-substituent on either phenyl ring has led to very potent inhibitors of the enzyme, the best being 4-phenylchalcone oxide (50% inhibition at 6.4 × 10−8m). Multiple factorial analysis on the inhibition data for the two series of chalcone oxides prepared (phenyl or benzoyl substituted) revealed both the essentialness of hydrophobic interactions and the apparent nonequivalence of the two hydrophobic sites involved in the inhibitory process. Steric factors were considerably less crucial while electronic effects were unimportant in the compounds examined. The chalcone oxides were either inactive or only weak inhibitors of the other major epoxide-metabolizing enzymes in mouse liver, cytosolic glutathione S-transferase, and microsomal epoxide hydrolase. The nature of the inhibition of cytosolic epoxide hydrolase by chalcone oxides was further investigated through steady-state kinetic analysis and the use of amino acid modifiers. Chalcone oxides give a slowly reversible mixed-noncompetitive inhibition. They may interact covalently with a cysteine residue possibly essential to the catalytic action of cytosolic epoxide hydrolase, and may indeed be alternative substrates with very low turnover. The cytosolic and microsomal epoxide hydrolases can be clearly distinguished by these inhibitors, further indicating different catalytic mechanisms.

Weighing Risk Factors Associated With Bee Colony Collapse Disorder by Classification and Regression Tree Analysis

ABSTRACT Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006–2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.

Neuroreceptor mechanisms in insect gustation: a pharmacological approach

Abstract Taste chemoreception is essential for animals to select suitable foods. Gustatory sensilla concentrated on mouthparts, other external appendages, or the food canal are responsible for transduction of chemical stimuli into nerve signals that trigger behavioral acceptance or rejection of a potential nutrient source. Insects have primary taste neurons containing both a dendrite and a direct axonal connection to the central nervous system, whereas receptor cells and afferent neurons are separated by a synapse in vertebrates. Taste receptor proteins have not been successfully purified or cloned from any animal to date. Our recent work with western corn rootworm beetles, Diabrotica virgifera virgifera LeConte, implicates a γ-aminobutyric acid (GABA)/glycine receptor in the perception of phago-stimulants and -deterrents. GABA stimulates feeding in herbivorous members of four orders of insects. The merits of this ligand-gated receptor model for chemoreception of ‘sweet’, ‘bitter’ and other taste classes will be contrasted with those proposed from vertebrate studies. Possibly one receptor gene family allows for insect perception of both food cues and potentially toxic non-host or environmental chemicals prior to their action at critical internal sites. Studies of taste receptors offer advantages over other insect neuroreceptors by their external location which simplifies ligand pharmacodynamics and allows coupled use of behavioral and electrophysiological methods to directly link receptor pharmacology with function.

Toxic and Behavioral Effects to Carabidae of Seed Treatments Used on Cry3Bb1- and Cry1Ab/c-Protected Corn

Abstract Most transgenic corn seed is now treated with systemic neonicotinoid insecticides. To address potential direct nontarget effects of these combined technologies, 16 Carabidae species from 10 genera (Agonum, Amara, Anisodactylus, Bembidion, Chlaenius, Harpalus, Patrobus, Poecilus, Pterostichus, and Scarites) field-collected from corn were directly exposed to Bacillus thuringiensis (Bt) Cry toxin-laden pollens and seed treatments in feeding and defined-dose bioassays. All adults readily fed on field or sweet corn pollens that expressed coleopteran-specific Cry3Bb1 or lepidopteran-targeting Cry1Ab/c, and no significant toxicity was observed. Adult survivorship ranged from 47 d for the predator Pterostichus melanarius (Illiger) to a year for the more omnivorous Scarites quadriceps Chaudoir, feeding solely on pollen containing 30–90 μg Cry3Bb1/g and water. In contrast, commercial doses of neonicotinoid seed treatments (imidacloprid, thiamethoxam, or clothianidin) elicited nearly complete mortality for 18 carabid species in 4-d bioassays containing corn seedlings. Carabid consumption of fungicide-only (fludioxonil plus mefenoxam) seed treatments was generally observed within 1 d, compared with a 2-d latency on neonicotinoid treatments, suggesting an antifeedant effect of the insecticide. In microcosm bioassays containing a corn seedling and five prey, clothianidin seed treatments killed adult western corn rootworm, Diabrotica virgifera virgifera LeConte and S. quadriceps, although the smaller Harpalus pensylvanicus (DeGeer) was more tolerant. We conclude that the neonicotinoid/fungicide seed treatments, and not Cry3Bb1 or CryIAb/c, are a major direct mortality factor for ground beetles. Field studies are needed to determine population and community level effects on Carabidae when these transgenic and seed-treatment technologies are combined.

Comparative Toxicities and Synergism of Apple Orchard Pesticides to Apis mellifera (L.) and Osmia cornifrons (Radoszkowski)

The topical toxicities of five commercial grade pesticides commonly sprayed in apple orchards were estimated on adult worker honey bees, Apis mellifera (L.) (Hymenoptera: Apidae) and Japanese orchard bees, Osmia cornifrons (Radoszkowski) (Hymenoptera: Megachilidae). The pesticides were acetamiprid (Assail 30SG), λ-cyhalothrin (Warrior II), dimethoate (Dimethoate 4EC), phosmet (Imidan 70W), and imidacloprid (Provado 1.6F). At least 5 doses of each chemical, diluted in distilled water, were applied to freshly-eclosed adult bees. Mortality was assessed after 48 hr. Dose-mortality regressions were analyzed by probit analysis to test the hypotheses of parallelism and equality by likelihood ratio tests. For A. mellifera, the decreasing order of toxicity at LD50 was imidacloprid, λ-cyhalothrin, dimethoate, phosmet, and acetamiprid. For O. cornifrons, the decreasing order of toxicity at LD50 was dimethoate, λ-cyhalothrin, imidacloprid, acetamiprid, and phosmet. Interaction of imidacloprid or acetamiprid with the fungicide fenbuconazole (Indar 2F) was also tested in a 1∶1 proportion for each species. Estimates of response parameters for each mixture component applied to each species were compared with dose-response data for each mixture in statistical tests of the hypothesis of independent joint action. For each mixture, the interaction of fenbuconazole (a material non-toxic to both species) was significant and positive along the entire line for the pesticide. Our results clearly show that responses of A. mellifera cannot be extrapolated to responses of O.cornifrons, and that synergism of neonicotinoid insecticides and fungicides occurs using formulated product in mixtures as they are commonly applied in apple orchards.

Learning Impairment in Honey Bees Caused by Agricultural Spray Adjuvants

Background Spray adjuvants are often applied to crops in conjunction with agricultural pesticides in order to boost the efficacy of the active ingredient(s). The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to spray adjuvants as well. It was previously unknown whether these agrochemicals have any deleterious effects on honey bee behavior. Methodology/Principal Findings An improved, automated version of the proboscis extension reflex (PER) assay with a high degree of trial-to-trial reproducibility was used to measure the olfactory learning ability of honey bees treated orally with sublethal doses of the most widely used spray adjuvants on almonds in the Central Valley of California. Three different adjuvant classes (nonionic surfactants, crop oil concentrates, and organosilicone surfactants) were investigated in this study. Learning was impaired after ingestion of 20 µg organosilicone surfactant, indicating harmful effects on honey bees caused by agrochemicals previously believed to be innocuous. Organosilicones were more active than the nonionic adjuvants, while the crop oil concentrates were inactive. Ingestion was required for the tested adjuvant to have an effect on learning, as exposure via antennal contact only induced no level of impairment. Conclusions/Significance A decrease in percent conditioned response after ingestion of organosilicone surfactants has been demonstrated here for the first time. Olfactory learning is important for foraging honey bees because it allows them to exploit the most productive floral resources in an area at any given time. Impairment of this learning ability may have serious implications for foraging efficiency at the colony level, as well as potentially many social interactions. Organosilicone spray adjuvants may therefore contribute to the ongoing global decline in honey bee health.