James L. Frazier

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.

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.

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.

Use of Feeding Inhibitors in Insect Control

The last 400 million years or so has seen a very complex process of coevolution between plants and phytophagous insects (Ehrlich and Raven, 1964; Futuyma and Keese, 1992). Plants, apart from creating physical and mechanical barriers, rely mostly on chemical protection against insect feeding. Numerous terpenoid, alkaloid, and phenolic feeding inhibitors have evolved in the course of this arms race between plants and insects (Harborne, 1993; Rosenthal and Berenbaum, 1992; Rosenthal and Janze, 1979). The proposed process is that a random mutation within the plant genome led to the synthesis of a new compound which prevented an insect from feeding on the plant. Those insects faced with strong selection pressures exerted by plant allelochemicals evolved a mechanism or mechanisms that allowed them to overcome the deterrent or toxic effects of the new compounds. In this way some defensive compounds were successfully counteracted by one or more insect species which allowed their use as unique feeding stimulants (e.g., cucurbitacins and diabroticite beetles); however, the phagodeterrent activity remained preserved against a great many other insects. The current list of phagodeterrents is very long and their potency varies greatly among insect species, suggesting that there are many highly diverse insect—plant relationships. It appears that phagodeterrents rather than nutrients are a major force in the development of present insect—plant interactions as was proposed more than 30 years ago by Thorsteinson (Thorsteinson, 1960) and has seen renewed emphasis (Berenbaum, 1986).

A scientific note on Varroa destructor found in East Africa; threat or opportunity?

In many areas of the world where it is managed, the honeybee, Apis mellifera, has been plagued by diseases, pests and parasites. Of these, the parasitic mite, Varroa destructor Anderson and Truman (Acari:Varroidae), is considered by many as the most devastating. We found this mite in honeybee colonies throughout Kenya and in Tanzania for the first time in early 2009. Beekeepers surveyed were neither aware of the mite’s presence nor had they observed any negative impact on the survival and/or productivity of their bees. In March of 2009, we sampled 38 honeybee colonies (likely A. m. scutellata, and possibly A. m. scutellata hybrids) in seven locations in Central and Eastern Kenya. We employed a common sampling technique to determine mite presence/absence that utilizes powdered sugar to dislodge mites from adult bees (Macedo et al., 2002). An average of 717 ± 43 bees per colony were sampled and Varroa mites were found in all 38 colonies examined with numbers ranging from 3–108 per sample and averaging 26.3 ± 25.9 per colony. In a further similar survey (April–May, 2009) of 125 additional colonies located in the eastern, western and coastal regions of Kenya (69 colonies in 18 locations), coastal Tanzania (18 colonies in 4 locations) including Ugunja and Pemba Islands, collectively referred to as Zanzibar (likely A.m. litorea), and Western Uganda (14 colonies in 4 locations), 87% of the

Identification of sensilla involved in taste mediation in adult western corn rootworm (Diabrotica virgifera virgifera LeConte).

A group of sensilla present on the maxillary galea of adult western corn rootworm,Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae) beetles has been identified morphologically and physiologically to be involved in taste mediation. There are approximately 15 chemosensory hairs on each galea. Bilateral removal of these structures resulted in a significantly reduced consumption of a strongly phagostimulant triterpenoid, cucurbitacin B, and led to increased ingestion of a phagodeterrent alkaloid, strychnine. Electrophysiological responses obtained via tip-recording of galeal chemosensilla with submillimolar concentrations of host and nonhost plant compounds resulted in dose responses overlapping with the effective behavioral ranges. Cucurbitacin B was found to evoke chemosensory responses at levels as low as 0.1µM. Sinceγ-aminobutyric acid (GABA) is an agonist. (-)-β-hydrastine and strychnine are antagonists, and cucurbitacin B has been proposed to act at a separate modulatory site of classical synaptic GABA and glycine receptor-channel complexes, results reported here raise the possibility that there are peripheral chemosensory receptor sites that may resemble, functionally and structurally, synaptic receptor sites in the central nervous system.

In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States

This study measured part of the in-hive pesticide exposome by analyzing residues from live in-hive bees, stored pollen, and wax in migratory colonies over time and compared exposure to colony health. We summarized the pesticide burden using three different additive methods: (1) the hazard quotient (HQ), an estimate of pesticide exposure risk, (2) the total number of pesticide residues, and (3) the number of relevant residues. Despite being simplistic, these models attempt to summarize potential risk from multiple contaminations in real-world contexts. Colonies performing pollination services were subject to increased pesticide exposure compared to honey-production and holding yards. We found clear links between an increase in the total number of products in wax and colony mortality. In particular, we found that fungicides with particular modes of action increased disproportionally in wax within colonies that died. The occurrence of queen events, a significant risk factor for colony health and productivity, was positively associated with all three proxies of pesticide exposure. While our exposome summation models do not fully capture the complexities of pesticide exposure, they nonetheless help elucidate their risks to colony health. Implementing and improving such models can help identify potential pesticide risks, permitting preventative actions to improve pollinator health.

An alternate route to insect pharmacophagy : The loose receptor hypothesis

The phagostimulatory response of some diabroticite cucumber beetles toward triterpene cucurbitacins is used as a model in support of an alternative hypothesis explaining the evolution of pharmacophagous feeding behavior in insects. Whereas the use of noxious compounds from nonhost sources for purposes other than nutrition or host-plant recognition (pharmacophagy) has historically been explained in terms of the ancestral host hypothesis, we suggest that the less than perfect specificity of the binding properties of some peripheral receptors provides an opportunity for novel compounds sharing the configuration and polarity of target molecules to elicit a feeding response by coincidence rather than adaptive design.

Assessing Honey Bee (Hymenoptera: Apidae) Foraging Populations and the Potential Impact of Pesticides on Eight U.S. Crops

ABSTRACT Beekeepers who use honey bees (Apis mellifera L.) for crop pollination services, or have colonies making honey on or in close proximity to agricultural crops, are concerned about the reductions of colony foragers and ultimate weakening of their colonies. Pesticide exposure is a potential factor in the loss of foragers. During 2009–2010, we assessed changes in the field force populations of 9–10 colonies at one location per crop on each of the eight crops by counting departing foragers leaving colonies at regular intervals during the respective crop blooming periods. The number of frames of adult bees was counted before and after bloom period. For pesticide analysis, we collected dead and dying bees near the hives, returning foragers, crop flowers, trapped pollen, and corn-flowers associated with the cotton crop. The number of departing foragers changed over time in all crops except almonds; general patterns in foraging activity included declines (cotton), noticeable peaks and declines (alfalfa, blueberries, cotton, corn, and pumpkins), and increases (apples and cantaloupes). The number of adult bee frames increased or remained stable in all crops except alfalfa and cotton. A total of 53 different pesticide residues were identified in samples collected across eight crops. Hazard quotients (HQ) were calculated for the combined residues for all crop-associated samples and separately for samples of dead and dying bees. A decrease in the number of departing foragers in cotton was one of the most substantial crop-associated impacts and presented the highest pesticide risk estimated by a summed pesticide residue HQ.