Honey bees (Apis mellifera L.; Hymenoptera, Apidae) are the most efficient pollinators in agroecosystems, responsible for the successful production of fruits, nuts, and vegetables, but they continue to face debilitating challenges. One of the major factors leading to these challenges could be linked to poor nutrition that results in weakening the colony, increasing susceptibility to pests and pathogens, and reducing the ability of bees to adapt to other abiotic stresses. Extensively used for commercial pollination, honey bee colonies regularly face exposure to limited diversity in their pollen diet as they are placed in flowering monocrops. Lack of access to diverse plant species compromises the availability of plant secondary compounds (phytochemicals), which, in small amounts, provide significant benefits to honey bee health. We analyzed the beneficial phytochemical content of honey and stored pollen (bee bread) samples from colonies in large apiaries through the active bee season. Samples were evaluated for 4 beneficial phytochemicals (caffeine, kaempferol, gallic acid, and p-coumaric acid), which have previously been shown to improve honey bee health. Our results, as relevant to the apiary locations in the study, indicated that p-coumaric acid is uniformly available throughout the season. Caffeine is completely absent, and gallic acid and kaempferol are not regularly available. Our results suggest the need to explore the potential to deliver beneficial phytochemicals as nutritional supplements to improve bee health. It may be vital for the pollination industry to consider such targeted dietary supplementation as beekeepers strive to meet the increasing demand for crop pollination services.

Honey bees continue to face challenges relating to the degradation of natural flowering habitats that limit their access to diverse floral resources. While it is known that nectar and pollen provide macronutrients, flowers also contain secondary metabolites (phytochemicals) that impart benefits including increased longevity, improved gut microbiome abundance, and pathogen tolerance. Our study aims to understand the role of phytochemicals in pesticide tolerance when worker bees were fed with sublethal doses (1 ppb and 10 ppb) of thiamethoxam (TMX), a neonicotinoid, in 20% (w/v) sugar solution supplemented with 25 ppm of phytochemicals-caffeine, kaempferol, gallic acid, or p-coumaric acid, previously shown to have beneficial impacts on bee health. The effect of phytochemical supplementation during pesticide exposure was context-dependent.With 1 ppbTMX, phytochemical supplementation increased longevity but at 10 ppb TMX, longevity was reduced suggesting a negative synergistic effect. Phytochemicals mixed with 1 ppb TMX increased mortality in bees of the forager-age group but with 10 ppb TMX, mortality of the inhive-age group increased, implying the possibility of accumulation effect in lower sublethal doses. Given that the phytochemical composition of pollen and nectar varies between plant species, we suggest that the negative impacts of agrochemicals on honey bees could vary based on the phytochemicals in pollen and nectar of that crop, and hence the effects may vary across crops. Analyzing the phytochemical composition for individual crops may be a necessary first step prior to determining the appropriate dosage of agrochemicals so that harm to bees Apis mellifera L. is minimized while crop pests are effectively controlled.

Continued loss of natural habitats with native prairies and wildflower patches is eliminating diverse sources of pollen, nectar and phytochemicals therein for foraging bees. The longstanding plant-pollinator mutualism reiterates the role of phytochemicals in sustaining plant-pollinator relationship and promoting honey bee health. We studied the effects of four phytochemicals-caffeine, gallic acid, kaempferol and p-coumaric acid, on survival and pathogen tolerance in the European honey bee, Apis mellifera (L.). We recorded longevity of worker bees that were provided ad libitum access to sugar solution supplemented with different concentrations of phytochemicals. We artificially infected worker bees with the protozoan parasite, Nosema ceranae. Infected bees were provided access to the same concentrations of the phytochemicals in the sugar solution, and their longevity and spore load at mortality were determined. Bees supplemented with dietary phytochemicals survived longer and lower concentrations were generally more beneficial. Dietary phytochemicals enabled bees to combat infection as seen by reduced spore-load at mortality. Many of the phytochemicals are plant defense compounds that pollinators have evolved to tolerate and derive benefits from. Our findings support the chemical bases of co-evolutionary interactions and reiterate the importance of diversity in floral nutrition sources to sustain healthy honey bee populations by strengthening the natural mutualistic relationships.

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is an important economic pest of maize (Zea mays L.) in North America and Europe. Previous efforts to formulate an artificial diet for western corn rootworm larvae highlighted an important role of corn root powder, which had a significant positive impact on several larval developmental traits. Unfortunately, this ingredient is not available for purchase. Toward the goal of developing an artificial diet for western corn rootworm larvae with all ingredients readily accessible, we conducted research to isolate essential growth factors for larval development from corn root powder to improve the performance of diet without corn root powder. For all experiments, multiple life history parameters (survival, weight, and molting) were recorded from 15-d diet bioassays. Corn roots may contain factors that assist in larval growth, but some of these factors were not fully extracted by methanol and remained in the extracted root. Methanolic extracts significantly increased molting to second instar, but did not significantly increase survival, dry weight, or molting to third instar, suggesting the primary corn root substituents affecting these factors cannot be extracted or other extraction methods may be required to extract the essential factors from corn roots. We showed that whole corn root powder was best when used in combination with all the other nutrient sources in the published western corn rootworm formulation. Corn root powder made from proprietary seed and Viking seed has similar value.

The susceptibility of western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae to nine insecticides from five different classes and to Bt proteins eCry3.1Ab and mCry3A in the presence or absence of feeding stimulants, was estimated in filter paper and diet toxicity assays, respectively. The use of a synthetic feeding stimulant blend of the sugars glucose, sucrose, and fructose plus linoleic acid at a ratio of 30:4:4:0.3 mg/ml of distilled water was evaluated to determine whether they increase the efficacy of insecticides and Bt proteins. The efficacy of thiamethoxam diluted in solutions with feeding stimulants was significantly increased when compared to thiamethoxam dilutions in water (>60-fold). Differences in the efficacy of the other insecticide classes when diluted in feeding stimulant solutions were no greater than fivefold when compared to the insecticides diluted in water. The presence of corn root juice as a natural feeding stimulant diminished toxicity of the insecticides, except for thiamethoxam, even though larval fresh weight was higher when fed on root juice compared to feeding stimulant or water. The use of feeding stimulants in diet toxicity assays did not enhance efficacy of eCry3.1Ab nor mCry3A proteins. Feeding stimulants can be recommended in combination with thiamethoxam to increase larval mortality. These results are discussed in terms of applicability of feeding stimulants to improve susceptibility of western corn rootworm larvae to pesticides in general.

Feeding behaviour, feeding intensity and staying behaviour of neonate western corn rootworm larvae ( Diabrotica virgifera virgifera LeConte) were evaluated in response to synthetic feeding stimulant blends to determine larval preferences among the three maize root sugars (glucose, fructose and sucrose) in the active blend and to determine whether any single sugar can substitute for the 3‐sugar combination in a feeding stimulant blend. These experiments demonstrated the strong affinity that western corn rootworm larvae have for the natural sugars found in maize roots and also showed that sucrose is the most preferred of the three primary maize root sugars. The blend containing sucrose at 30 mg/ml elicited feeding that was not significantly different than the natural glucose:fructose:sucrose blend. In subtraction bioassays, removal of sucrose from the blend resulted in significantly fewer larvae feeding. When the three‐sugar blend was substituted with one of the number of various mono‐, di‐ or trisaccharides, fewer larvae fed on all of the treatments compared to the blend with sucrose, except for the blend with maltose. In feeding choice tests, larvae preferred a blend containing sucrose over blends with either glucose or fructose, but larvae chose equally between a blend with sucrose and a blend containing the three‐sugar mixture found in maize roots. Based on these results, a feeding stimulant blend with glucose (30 mg/ml), fructose (4 mg/ml) and sucrose (4 mg/ml) elicits the strongest feeding response, but sucrose alone, in amounts equivalent to the total maize root sugar concentration (30 mg/ml), could serve as a substitute for the 3‐sugar mixture in a synthetic feeding stimulant blend.

Methyl anthranilate (MA), a compound in maize roots that is repellent to western corn rootworm larvae (Diabrotica virgifera virgifera LeConte) was tested in behavioral bioassays in a soil environment. MA prevented larvae from locating roots of a maize seedling, and the repellency strengthened with increasing rates of MA. In a simple pushpull strategy between an MA-treated seedling and an untreated seedling, granules containing 0.1 mg/g MA pushed larvae to the untreated seedling. This push effect increased with dose, with 90% repellency observed for the highest dose tested (100 mg/g). Chemical analysis showed that MA concentrations remained high for 4 wk in dry, sterilized or unsterilized soil, but declined rapidly in moist soil. After 7 d, 50% less MA was recovered in moist, sterilized soil than in dry soil, and only a trace of MA remained in unsterilized moist soil, suggesting that both moisture and microbial activity contributed to the loss of MA. Various (MA) carrier granules were tested in bioassays after aging in moist soil. After 1 d, all of the MA granules were repellent at the 10 mg/g rate and clay granules were also effective at 1 mg/g. After 1 wk, only molecular sieve granules elicited repellency, but that activity disappeared after 2 wk. These results demonstrate that MA is repellent to western corn rootworm larvae in the soil environment and may have potential as a rootworm treatment if formulations can be developed that protect the material from decomposition in the soil.

Pollen is essential for successful plant reproduction and critical for plant-pollinator mutualisms, as pollen is essential larval nutrition. However, we understand very little about the chemical constituents of pollen leading us to this exploratory study characterizing plant and beehive pollen.

s of Poster Presentations 44. Using ‘omics’ tools to breed better Canadian bees Renata S. Borba1,2, Robert Currie3, Nicolas Derome4, Pierre Giovenazzo5, Marta Guarna2, Shelley Hoover6, Amro Zayed7, Leonard J. Foster1, Stephen F. Pernal2. 1University of British Columbia, Center for High-Throughput Biology, Vancouver, BC, Canada. 2Agriculture and Agri-Food Canada, Beaverlodge Research Farm, Beaverlodge, AB, Canada. 3Department of Entomology, University of Manitoba, Winnipeg, MB, Canada. 4Institut de Biologie Intégrative et des Systèmes, Université Laval, Québec, QC, Canada. 5Département de Biologie, Université Laval, Québec, QC, Canada. 6Alberta Agriculture and Forestry, Lethbridge, AB, Canada. 7Department of Biology, York University, Toronto, ON, Canada. Overwintering mortality of honey bee colonies in Canada has been continuously greater than the acceptable range of 0% to 15% since the winter of 2006/2007. The main causes of colony death, as reported by Canadian beekeepers, include high pathogen/parasite infestation levels (e.g., Varroa mites, Nosema spp.), poor quality queens and severe weather conditions. Every year, Canadian beekeepers import hundreds of thousands of queens, mainly from the U.S.A. and New Zealand. The importation of foreign queens has the potential to introduce undesirable pathogens or genetics and supply bees that have not been selected to survive in northern temperate climates. The goal of our project is to measure 12 economically-valuable traits of honey bees (colony phenotypes) and develop genomic and proteomic markers for each trait that will enable beekeepers to rapidly select and breed healthy and productive colonies that are well adapted to the Canadian climate. In 2016, 1025 colonies across Canada (British Columbia, Alberta, Manitoba, Ontario and Quebec) were sampled and phenotypic data was collected for the following colony-level traits: 1) Varroa mite population growth 2–4) grooming, hygienic and defensive behaviour; 5) honey production; 6) sealed brood population; 7–9) pathogen abundance (viruses, Nosema spp., Trypanosomatids); 10) innate immunity factors; 11) gut microbiota; and 12) overwintering success. The identification of bio-markers for each trait, and the variation of each trait among colonies located in different landscapes and climates in Canada, as well as the correlation between phenotypes comprised the first step of this novel research. To date, we have performed multiple correlation analysis to study the relationship among all traits. The two highest correlation values found are from between fall and spring total gross colony weights, and total and instantaneous honey production (see Figure 22). Colony weight in the spring (after winter) is highly correlated with its weight in the fall (before winter; R2 = 0.8374; P < 0.001). We also found that total honey production is highly correlated with total colony weight gain during 2 weeks of peak honey flow (R2 = 0.6905; P < 0.001). This result is supported by previous research from T. Szabo who found that 86% of the net honey production occurs in 14 days during the peak of honey flow (Szabo T and Heikel D, 1987 J. Apic. Res., 26(1): 47–52). In the summer of 2017, 496 colonies were sampled and putative markers were validated against a test population, with the end goal of having this technology transferred to end-users, such as the National Bee Diagnostic Centre (Beaverlodge, AB), where it will be made available to beekeepers. This is the first large-scale study for marker assisted selection in honey bees using integrated genomics and proteomics tools. Our innovative research will promote a healthier honey bee population and support the sustainability of the Canadian beekeeping industry.

Conifer defenses against bark beetle attack include, but are not limited to, quantitative and qualitative defenses produced prior to attack. Our objective was to assess host defenses of lodgepole pine and ponderosa pine from ecotone stands. These stands provide a transition of host species for mountain pine beetle (Dendroctonus ponderosae; MPB). We asked two questions: (1) do the preformed quantitative host defenses (amount of resin) and (2) the preformed qualitative host defenses (monoterpene constituents) differ between lodgepole and ponderosa pines. We collected oleoresins at three locations in the Southern Rocky Mountains from 56 pairs of the pine species of similar size and growing conditions. The amount of preformed-ponderosa pine oleoresins exuded in 24 h (mg) was almost four times that of lodgepole pine. Total qualitative preformed monoterpenes did not differ between the two hosts, though we found differences in all but three monoterpenes. No differences were detected in α-pinene, γ-terpinene, and bornyl acetate. We found greater concentrations of limonene, β-phellandrene, and cymene in lodgepole pines, whereas β-pinene, 3-carene, myrcene, and terpinolene were greater in ponderosa pine. Although we found differences both in quantitative and qualitative preformed oleoresin defenses, the ecological relevance of these differences to bark beetle susceptibility have not been fully tested.

Feeding behaviour, feeding intensity and staying behaviour of neonate western corn rootworm ( Diabrotica virgifera virgifera LeConte) larvae were evaluated in response to synthetic feeding stimulant blends. All of the treatments contained a 3‐sugar blend (glucose : fructose : sucrose, 30 : 4 : 4 mg/ml) and one of twelve free fatty acids. Each free fatty acid was tested in this blend at three different concentrations. The addition of the 12 : 0, 16 : 0, 16 : 1, 18 : 0, 18 : 1, 18 : 2 and 18 : 3 free fatty acids to the sugar blend significantly (P < 0.05) increased the percentage of larvae feeding, but did not increase food consumption per larva. Most of the free fatty acids elicited staying behaviour. At the lowest dose (0.1 mg/ml), all of the free fatty acids except the 18 : 0 and the 20 : 0 elicited staying by significantly more larvae than the sugar blend, and at the highest dose (1.0 mg/ml), eight free fatty acids (8 : 0, 10 : 0, 12 : 0, 14 : 0, 16 : 1, 18 : 1, 18 : 2 and 18 : 3) caused more larvae to stay compared to the sugar blend. Larvae were visibly impaired after exposure to some of the free fatty acids. At the highest dose, the 8 : 0, 10 : 0, 12 : 0, 14 : 0, 16 : 1, 18 : 1 and 18 : 2 free fatty acids were toxic to the larvae. At least 60% of larvae were impaired after exposure to the 12 : 0, 16 : 1 and 18 : 2 free fatty acids and the 8 : 0 and 10 : 0 free fatty acids caused 100% impairment or death. Synthetic blends were compared with liquid from crushed maize roots and with a methanol extract of maize roots. Feeding intensity and staying behaviour on the root liquid and the root extract were significantly greater than on any of the synthetic blends, suggesting the presence of additional compounds in maize roots that serve as feeding cues for western corn rootworm larvae.

Similar to aboveground herbivores, root-feeding insects must locate and identify suitable resources. In the darkness of soil, they mainly rely on root chemical exudations and, therefore, have evolved specific behaviours. Because of their impact on crop yield, most of our knowledge in belowground chemical ecology is biased towards soil-dwelling insect pests. Yet, the increasing literature on volatile-mediated interactions in the ground underpins the great importance of chemical signalling in this ecosystem and its potential in pest control. Here, we explore the ecology and physiology of these chemically based interactions. An evolutionary approach reveals interesting patterns in the response of insects to particular classes of volatile or water-soluble organic compounds commonly emitted by roots. Food web analyses reasonably support that volatiles are used as long-range cues whereas water-soluble molecules serve in host acceptance/rejection by the insect; however, data are still scarce. As a case study, the chemical ecology of Diabrotica virgifera virgifera is discussed and applications of belowground signalling in pest management are examined. Soil chemical ecology is an expanding field of research and will certainly be a hub of our understanding of soil communities and subsequently of the management of belowground ecosystem services.

Behavioral bioassays were used to isolate compounds from germinating corn roots that elicit a host recognition response (tight-turning behavior) by neonate larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte. When a behaviorally active extract of germinating corn roots was separated into an aqueous partition and a hexane partition, significantly more larvae (P < 0.05) responded to the recombined partitions than to either partition alone, demonstrating that the active material is a blend comprising both polar and nonpolar compounds. When the aqueous partition was separated with reverse-phase solid phase extraction, most of the behavioral activity was retained in the 100% water fraction (F-1). Gas chromatography-mass spectrometry analysis determined that F-1 contained a blend of small sugars, diacids, amino acids, and inorganic compounds. The nonpolar partition was separated on a silica column, and the resulting fractions were tested in combination with F-1 from the aqueous separation. More than 70% of larvae responded to the 100% acetone fraction (fraction B) in combination with F-1, and the response to this treatment was significantly higher than responses to the other nonpolar fractions or to F-1 alone. Methyl esterification of fraction B, followed by gas chromatographic fatty acid methyl ester analysis, confirmed that fraction B primarily consisted of lipids containing fatty acyl groups.

In bioassays, the addition of a synthetic feeding stimulant blend improved the efficacy of the insecticide thiamethoxam against neonate western corn rootworm, Diabrotica virgifera virgifera LeConte, larvae. In 4‐h bioassays, the concentration of thiamethoxam required for 50% mortality (LC50) was 2800 pg/ml for the insecticide alone, but was reduced to 0.0075 pg/ml when the synthetic feeding stimulant blend was added (more than a 100,000‐fold difference). Dilutions of thiamethoxam and tefluthrin (ranging from 0 pg/ml to 10 μg/ml) were tested in behavioural bioassays alone and in conjunction with a feeding stimulant blend containing 30 : 4 : 4 : 1 mg/ml glucose : fructose : sucrose : linoleic acid. Larvae were placed on insecticide‐treated filter paper disks in an arena that allowed them to leave the treated area at will. In 30‐min bioassays, and in 4‐h bioassays, significantly more larvae fed on thiamethoxam‐treated disks when the feeding stimulant blend was present for every concentration of insecticide tested. Larvae fed readily on disks treated with tefluthrin when the feeding stimulants were added, but the feeding stimulant blend did not affect the activity of tefluthrin insecticide at any concentration tested.

SUM2162 is the first known example of a naturally occurring maize, Zea mays L., genotype with antixenosis (nonpreference) resistance to western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), larval feeding. Behavioral responses of neonate western corn rootworm larvae were evaluated in laboratory bioassays with seven maize genotypes selected for native resistance to rootworm feeding damage. Two susceptible maize genotypes and one transgenic (Bacillus thuringiensis) maize genotype were included as controls. In soil bioassays with cut roots, no larvae entered the roots of the resistant variety SUM2162, but at least 75% of the larvae entered the roots of every other maize type. Larvae made significantly fewer feeding holes in the roots of SUM2162 than in all the other maize genotypes, except the isoline control. In feeding bioassays, larval feeding varied significantly among maize genotypes, but there was no significant difference between the resistant varieties and the susceptible controls. There were no significant differences among any of the genotypes in host recognition (search) behavior of larvae after exposure to the roots. Little variation in feeding stimulant blends was observed among maize genotypes, indicating minimal contribution to the observed antixenosis.

Cues that elicit a characteristic localized search behavior by neonate larvae of the western corn rootworm. Diabrotica virgifera virgifera Leconle (Coleoptera: Chrysomelidae), were extracted from living corn Zea mays I roots with acetone. Larvae were exposed to corn roots or to an acetone extract of corn roots and then transferred into a bioassay arena where their movements were tracked and recorded. After a 5-min exposure to live corn roots, larvae produced highly convoluted tracks that were indicative of a localized search behavior and these distinctive tracks were also produced by larvae exposed to an acetone extract of corn roots, larvae exposed to filter paper control moved in relatively straight paths that were indicative of ranging behavior. Larval tracks were recorded by means of a video camera and tracking software and four parameters of movement were used to quantify the tracks mean turn angle, mean meander total distance and maximum distance from origin. For every parameter measured, tracks resulting from exposure to the control were significantly different from tracks resulting from exposure to live corn roots and to all doses of the corn root extract. In a separate experiment, larvae exposed to corn root pieces or corn root juice exhibited the localized search behavior, but larvae exposed to oat root pieces and oat root juice (nonhost) exhibited ranging behavior.

Termite bait stations with orwithout a CO 2 -generatingbait (Focus Termite Attractant, produced by Brotica, Inc., Fort Collins, Colorado, and distributed by Ensystex Australasia) were tested at tree and house locations in Australia. The termite species Coptotermes acinaciformis (Froggatt) (Isoptera: Rhinotermitidae), Schedorhinotermes intermedius (Brauer) (Isoptera: Rhinotermitidae), Microcerotermes turneri (Froggatt) (Isoptera: Termitidae), and Nasutitermes exitiosus (Hill) (Isoptera: Termitidae) discovered more monitoring stations when the CO 2 -generating bait was present, and also discovered the monitoring stations more quickly when the CO 2 -generating bait was present.

Amounts of the insecticide thiamethoxam required for 50% mortality of western corn rootworm larvae, Diabrotica virgifera virgifera LeConte, were reduced 100-fold when extracts of germinating corn, Zea mays L., were used to entice neonate larvae to feed on it. In behavioral bioassays, neonate rootworm larvae fed vigorously on filter paper disks treated with liquid pressed from corn roots. Moreover, disks treated with an acetone extract of corn (dried and rewetted with water) also elicited strong feeding from larvae. Larvae wandered away from filter paper disks treated with distilled water without feeding. Dilutions of thiamethoxam were tested in the bioassay alone or with corn extract and the efficacy of this insecticide was improved by the addition of the corn extract. For solutions containing 10 ppm thiamethoxam, 95% larval mortality occurred after 30 min of exposure when corn extract was present, but only 38% mortality occurred when the same concentration of insecticide alone (no feeding stimulants) was tested. Larval mortality after 24 h was significantly higher for corn extract-treated disks with 0.01, 0.1, 1, or 10 ppm insecticide than for the same concentrations without corn extract. Thiamethoxam did not deter larval feeding on corn extract, even at the highest concentration of thiamethoxam tested.