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American Bee Journal Extra

January 18, 2013

Proceedings of American Bee

Research Conference,

Hershey, PA, 2013

Compiled/edited by

Zachary Huang

aDepartment of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand (e-mail:, bDepartment of Basic Science and Craniofacial Biology, New York University College of Dentistry, NY, 10010, USA,cMaterials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
    Asian giant honey bees are vital honey produces and pollinators of cultivated crops and wild plants. Relationships between gut microorganisms and honey bees are essential for maintaining proper nutrition and immunity. This study examined the bacterial community structures in the midgut of the Asian giant honey (A. dorsata) workers collected from two locations in Chiang Mai, Thailand. A total of 180 workers from six colonies were collected at different geographic sites in northern Thailand.  Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) is a cultivation-independent molecular fingerprinting technique that allows the assessment of the predominant bacteria species present in bee midguts. The result showed the mean species richness and the Shannon index differed between colonies but nor by locations. Bacterial DNA profiles had similar patterns in individual colonies which differed amongst the replicate colonies, but was not affected by graphical location. Sequence analysis of DGGE products revealed evidence for core bacteria of the genera Gammaproteobacteria and Firmicutes. Although core bacteria existed in both populations, specific bacterial species were observed for each colony and site.
2. Milbrath, M.O.a, X.B. Xiea, b, & Z. Y. Huanga  CARBON DIOXIDE ANESTHESIA AFFECTS MORTALITY OF NOSEMA CERANAE INFECTED HONEY  BEES aDepartment of Entomology, Natural Science Building, 288 Farm Lane Room 243, Michigan State University, East Lansing, MI 48824, USA,
bDepartment of Laboratory Animal Science, 71 East Yangming Road, Nanchang University, Nanchang, Jiangxi 330006, China
    We have known for almost a century that the microsporidia Nosema apis is a serious parasite of the Western honey bee (Apis mellifera).  Only recently, we have identified that a related microsporida, Nosema ceranae, has transferred from its original host, the Eastern honey bee, and is causing serious infection in A. mellifera as well.  The full effects of Nosema ceranae infection in this new host remain unknown.  Numerous studies have examined mortality after experimental infection with N. ceranae, but they have had highly variable results. One reason for this variation may be differences in experimental techniques. We examined one technique, CO2 anesthesia, that may affect honey bee survival in the presence of nosema infection. We hypothesized that the use of CO2 anesthesia when infecting bees would reduce survival.  We used four treatments (Control, Nosema only, CO2 anesthesia only, CO2 anesthesia /Nosema), repeating the experiment with three colonies.  We found that bees infected with Nosema spores alone had significantly lower survival than control bees (median survival = 21 days and 23 days, respectively), and that CO2 anesthesia had a greater effect on survival than nosema infection alone. Bees infected using CO2 anesthesia survived for significantly shorter times, regardless of their infection status (median survival = 18 days for both groups). Interestingly, bees infected using CO2 had significantly fewer spores than bees infected without anesthesia.  These results indicate differences in honey bee mortality experiments may be due in part to experimental technique. Overall, our survival rates were higher than these previous nosema mortality experiments, indicating that variation in honey bee resistance to nosema may be an important factor in determining survival after being infected with this parasite.
3. Xie, X.B.a, b, G.W. Bianb, Z. Xib, & Z.Y. Huangb-USING RNAi TO HUNT FOR GENES IMPORTANT FOR VARROA SURVIVAL AND REPRODUCTION.aLaboratory Animal Science Department, Nanchang University, Nanchang, Jiangxi, 330006, China, bDepartment of Entomology, 243 Natural Science, Michigan State University, E. Lansing, MI, 48824, USA
    The varroa mite, Varroa destructor, is the worst pest of the Western honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide. In this study we used RNA interference (RNAi) technology to disrupt the life cycle of varroa mites by either causing death or causing a reduction in reproduction. We searched for gene orthologs in the newly established varroa mite genome ( We tested the genes of Daughterless (Da), Proteasome 26S subunit 4 (Pros26.4), Ribosomal protein L8 (RpL8), Ribosomal protein L11 (RpL11), Ribosomal protein P0 (RpP0), and Ribosomal protein S13 (RpS13), all of which have shown to play roles in survival or reproduction in other tick species.
    Results showed that our method of microinjection worked well because the survival of 48-h post injection (p.i.) was 85.51 ± 1.98 % (mean ± SE) for GFP injected groups. Gene suppression efficiency at 48-h pi was 62~84% for four of the genes we tested. After microinjection, we assessed the effects on mite survival of 2 and reproduction of 4 candidate genes: Pts26.4 gene and Da gene caused a significantly reduction in mite survival compared to the GFP control. For the other four genes, no significant effect on survival was observed so we tested the effect of dsRNA on mite reproduction. The mean (± SE) number of female offspring of mites injected with dsRNA of RPL8, RPL11, RPP0, and RPS13 were 1.51 ± 0.20 (N=146), 0.20 ± 0.10 (N=94) and 1.05 ± 0.09 (N=90) and 1.30 ± 0.18 (N= 129), respectively.  All these were statistically significantly lower compared to their own GPF injected controls (T-test, P < 0.001 for each gene). RPL8, RPL11, RPP0 and RPS13 therefore seem to affect reproduction in Varroa destructor.
    In conclusion, we have discovered four genes important for mite reproduction and two genes important for mite survival. Future goals are to find ways to introduce these genes into varroa mites so that their survival or reproduction can be suppressed.
4. Guzman-Novoa, E., G. Koleoglu, K. Reyes-Quintana & P.H. Goodwin – CELLULAR IMMUNE RESPONSE TO VARROA MITE INFESTATION IN EUROPEAN AND AFRICANIZED BEES School of Environmental Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada
 Varroa destructor feeds on the haemolymph of the honey bee, leaving an open wound in its host. Wound healing would be a consequence of a cellular immune response by the infested bee. However, not much is known about how Varroa affects the haemocyte response of bees to its infestation over time, and even less if different genotypes of bees have similar responses to the parasite.
     Newly emerged Africanized and European honey bees were artificially infested with varroa mites, were punctured with an entomological pin, were injected with a macerate of varroa mites or with the buffer used for the macerate, and were compared with control, untreated bees, for their cellular immune response. Haemocyte counts were obtained from bees sampled at different time points. Piercing resulted in a rapid (2 hour) increase in number of haemocytes in the haemolymph of bees of both types, indicating a response to heal the wound. However, when bees were infested with mites or injected with varroa macerate, the response in haemocyte numbers decreased significantly within 12 hours relative to the control or buffer injection treatments in both types of bees. These results suggest that Varroa inoculates components that inhibit the cellular response of Africanized and European honey bees, possible through saliva secretions.
5. Medrzycki, P.a, S. Tosia,b, G. Bogoa, & C. Porrinib - INFLUENCE OF TEMPERATURE ON HONEY BEE SUSCEPTIBILITY TO PESTICIDES aCRA-API (Consiglio per la Ricerca e la Sperimentazione in Agricoltura; Unità di ricerca di apicoltura e bachicoltura), 80, Via di Saliceto, Bologna, 40128, Italy, bDepartment of Agricultural Sciences, University of Bologna, 44, Via Fanin, Bologna, 40127, Italy
     EPPO (2010 EPPO Bulletin 40(3): 313-319) and OECD (1998) are the European official guidelines that describe how to conduct trials for the evaluation of side-effects of plant protection products on honey bees. According to these guidelines, acute oral toxicity tests on adult honey bees should be carried out at 25±2°C.
    In nature, adult forager bees may be exposed to a wide range of temperatures: from about 15°C (when foragers fly in spring) to 35°C (brood nest temperature) or even more (outside in hot climates). Since forager bees may also be exposed to pesticides, the purpose of this work was to investigate the influence of temperature on the susceptibility of forager bees to these substances.
    Exiting forager bees from healthy and queen-right colonies were collected. Subsequently, acute oral LD50 tests were carried out at three different temperatures: 25±0.5, 30±0.5 and 35±0.5°C. Three active ingredients (fipronil, clothianidin and thiamethoxam) were separately tested. Following the European official guidelines (EPPO and OECD), five different doses of each a.i. and a control were provided via bulk administration in 10μL 50% w/w of sucrose solution per bee. Three or four replicates in different seasons were carried out. Mortality at 24 hours was assessed and LD50 with confidence intervals were calculated (Probit analysis, Polo LeOra software).
    The results show that the LD50 value depends on the test temperature. This relationship was confirmed statistically in all the replicates of fipronil and thiamethoxam and in 2 of 4 replicates of clothianidin. Furthermore, different substance groups have different LD50 trends in relation to the temperature. In fact, with the increase of the temperature A) the toxicity of fipronil (phenylpyrazole) increases, while B) the toxicities of clothianidin and thiamethoxam (neonicotinoids) decrease.
    To conclude, the toxicity of pesticides to forager bees is influenced by the temperature which the bees are exposed to. Interestingly, the strength and sign of this correlation depend on the characteristics of the a.i./substance group.
    The European official guidelines used in the pesticide registration process (EPPO, OECD) allow to perform toxicity tests at a single temperature within 23-27°C: this wide range gives the interested parts the opportunity to carry out the tests at the temperature that will cause the preferred effect. Thus, to carefully evaluate the effects of an a.i., toxicity tests should be carried out at least at two different temperatures distant by 10°C (e.g. 25°C and 35°C). Otherwise, the hazard which the bees are exposed to could be underestimated.
6. Tosi, S.a,b, D. Bergaminia, C. Porrinia & P. Medrzyckib- INFLUENCE OF POLLEN QUALITY ON HONEY BEE HEALTH aDepartment of Agricultural Sciences, University of Bologna, 44, Via Fanin, Bologna, 40127, Italy, bCRA-API (Consiglio per la Ricerca e la Sperimentazione in Agricoltura; Unità di ricerca di apicoltura e bachicoltura), 80, Via di Saliceto, Bologna, 40128, Italy
    It is commonly agreed that the phenomenon named CCD (Colony Collapse Disorder), related to the recent honey bee colony losses, is multi-factorial. One of the factors, suspected of playing an important role in these losses, is the nutritional status of the colonies (Oldroyd, 2007 PLoS Biol. 5(6):e168; vanEngelsdorp & Meixner, 2010 J Invertebr. Pathol. 103:80-95).
    Honey bees need to eat pollen to ensure the proper development and growth. Indeed, pollen is their main source of proteins. Forager bees tend to collect pollen from different plant species (Dimou & Thrasyvoulou, 2009 Apidologie 40:124-133) and this behavior helps to completely satisfy the nutritional requirements of the colony through a balanced and varied diet (Brodschneider & Crailsheim, 2010 Apidologie, 41(3):278-294). In fact, the relative proportion of the nutrients in the pollen can vary widely according to its botanical origin. Nevertheless, commercial colonies are often placed in agricultural landscapes, where usually there are few pollen-producing plant species available for the bees. For this reason, forager bees can collect pollen from only few different plant species available, according to the flowering time.
    The aim of this work was to investigate if the quality of the pollen available to a colony can influence the health of the bees.
     Fresh pollen loads from apiaries situated either in natural (NAT) or intensive agriculture (AGR) ecosystems were collected. The AGR pollen was characterized by lower diversity of botanical origin and lower protein content than NAT pollen. No insecticide residues were found in the tested pollen.
    Newly emerged bees from the same healthy and queen-right colony were collected. Then, the bees were incubated in laboratory at 30°C and fed with water, organic Robinia honey and fresh pollen (AGR or NAT) ad libitum. Mortality and food consumption during incubation were assessed.
    After 2 weeks of incubation, acute oral LD50 tests were carried out. Two active ingredients (fipronil and thiamethoxam) were separately tested. Six test doses including control were administered to the bees, through bulk administration of an a.i. in 10μL 50% w/w sucrose solution per bee. Honey bee mortality was assessed at 24, 48, 72 hours. LD50 and its confidence intervals were calculated (Probit analysis, Polo LeOra software).
    The results showed that bees fed with AGR pollen, compared to those fed with NAT pollen, were characterized by: 1) higher mortality during the 2 weeks of incubation and 2) lower resistance to the intoxication by fipronil. No significant effect of the pollen quality on the susceptibility of the bees to thiamethoxam was found. In addition, more NAT pollen than AGR pollen was consumed by the bees during the incubation period.
    To conclude, the survival of the bees and their susceptibility to pesticides may be influenced by the pollen nourishment. In this case study, Italian pollen with low diversity of botanical origin and low protein content (AGR) reduced the longevity of the bees and their resistance to fipronil (phenylpyrazole) but not to thiamethoxam (neonicotinoid). Thus, intensive agricultural landscapes may have negative impact on honey bee colonies through both the widespread presence of pesticides and the low nutritional quality of the pollen available.
     Finally, it is assumed that the same stressor (e.g. intoxication) can cause lower or greater consequences to the colony in relation to the pollen supply/location of the colony.
7. Mullin, C.A., J. Chen, W. Zhu, M.T. Frazier & J.L. Frazier - THE FORMULATION MAKES THE BEE POISON - Department of Entomology, Center for Pollinator Research, The Pennsylvania State University, University Park, PA 16802
    Modern pesticide formulations, particularly when multiple active ingredients are blended, require proprietary adjuvants and ‘inerts’ to achieve high efficacy for targeted pests. Although numerous pesticides have been found in beehive samples, no individual pesticide amount correlates with recent bee declines. Formulations usually contain inerts at higher amounts than active ingredients, and these penetrating enhancers, surfactants and adjuvants can be more toxic on non-targets than the active ingredients. For example, we found that the miticide formulation Taktic® was four time more orally toxic to adult honey bees than the respective active ingredient amitraz. Impacts of 'inerts' in pollen and nectar alone or in combination with coincident pesticide residues on honey bee survival and behavior are unknown. An improved, automated version of the proboscis extension reflex 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. Learning was impaired after ingestion of 20 µg of any of the four tested organosilicone adjuvants, 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.
    Monitoring methods are needed for major adjuvant residues so risks of formulation additives and their pesticide synergisms for pollinators can be assessed. Organosiloxane, nonyl- and octyl-phenol polyethoxylates are widely used as nonionic surfactants around honey bee hives or in their foraging areas as spray adjuvants or additives in agrochemical formulations. A method for analysis of organosiloxane, nonylphenol and octylphenol polyethoxylate surfactants in bee hive matrices was developed. A combined liquid-liquid extraction and solid phase extraction method was used. Less than 2 grams of honey, pollen or wax were extracted using the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach. Identification and quantification were accomplished employing liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS). Nonylphenol more than organosiloxane and octylphenol polyethoxylates were found in wax samples, while pollen and particularly honey residues were lower. We will continue to focus on recent formulation technologies, including organosilicone surfactants and solvents like N-methylpyrrolidone (NMP), of unknown bee ecotoxicity, and to investigate the possibility of recent bee declines being associated with these ‘inerts’.
    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 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. NMP was more orally toxic to larvae than adult honey bees. We have shown that chronic dietary exposure to a fungicide, pesticide mixtures, and a formulation ingredient have the potential to impact honey bee populations, and warrants further investigation.
8. Johnson, R. & E. Percel. The EFFECTS OF THE FUNGICIDE PRISTINE ON QUEEN REARING.  The Ohio State University, Department of Entomology, Wooster, OH
    There have been reports by commercial queen producers of occasional unexplained loss of large numbers of developing queens in the larval or pupal stage.  Many of the affected queen-rearing operations are situated among the almond orchards of California and report these losses in the weeks after almond trees bloom.  Almond flowers are a rich foraging resource for bees, but are also commonly treated with fungicides, insecticides and spray adjuvants during bloom to control pests and pathogens.  Queen producers have associated queen developmental problems with application of the fungicide Pristine, which contains the active incredients boscalid and pyraclostrobin, and the spray adjuvants containing organosilicone compounds.  To test the effect of these pesticides queens were reared in closed swarm boxes for four days, until capping, with nurse bees fed pollen treated with four concentrations of Pristine (0.4, 4, 40 and 400 ppm), an organosilicone-containing spray adjuvant (Break-Thru, 200 ppm), the combination of Pristine and Break-Thru (400: 200 ppm), diflubenzuron (100 ppm) as a positive control or water as negative control.   Low concentrations of pyraclostrobin (50 ppb), but no boscalid, was detectable in royal jelly fed to queens in the 400 ppm Pristine treatment.  No significant difference in queen survival to capping or adult queen emergence was observed between any of the experimental treatments and the negative control. Only diflubenzuron, the positive control, caused a substantial reduction in queen cell capping.  Interestingly, diflubenzuron use in almonds during bloom, at roughly the same time and scale as Pristine application, has seen on a steady increase over the last decade.  Future work should focus on the role of diflubenzuron, possibly in combination with other pesticides, on queen development, survival and success.
9. Villar, G., T. Baker, H. Patch, & C.M. Grozinger – EXAMINING THE CAUSES OF DIFFERENTIAL RESPONSES TO THE QUEEN BY DRONES AND WORKERS. Pennsylvania State University, Chemical Ecology Lab – Room 101, University Park, PA 16802
    In honey bees, the social interactions of workers and drones with the queen are primarily mediated by pheromones. Of these, a unique component of the queen mandibular gland pheromone, 9-ODA, has been found to function as both a social and sex pheromone. In workers, 9-ODA serves as an attractant, signaling the queen’s presence in the hive and playing a role in the formation of the queen’s retinue (Boch et al., 1975 J Chem. Ecol. 1:1:133-148). It also inhibits new queen rearing, slows worker maturation, and alters brain gene expression (Grozinger et al., 2003 PNAS 100:2). In drones, its effects are less well characterized, though we know it serves as a long range attractant, which allows drones to locate reproductively receptive queens at aerial congregation mating sites (Boch et al., 1975 J Chem. Ecol. 1:1:133-148).
     The role that 9-ODA plays for the members of the hive is developmentally and spatially context dependent, however. Drones show no attraction to the queen while inside a hive, at any age. They also don’t take mating flights outside of the hive before reaching maturity (Giray and Robinson, 1996 PNAS 93:21). Even after reaching sexual maturity, drones only attempt to find and mate with queens at specific times during the day. Workers, by contrast, are receptive to the queen soon after emerging; this is when they participate in queen tending and rearing and it is at this time that attraction to the queen and to 9-ODA is strongest. As workers age and transition from nurses to foragers, exposure and receptivity to the queen decrease. Though we have a good sense of the contextual dependency of the behavioral interactions with the queen in workers and drones, our understanding of the physiological and molecular mechanisms underlying these differential behaviors is not well understood.
    Here I discuss several studies which look at physiological and molecular phenomena that may be modulating worker and drone behavioral receptivity to the queen pheromone component, 9-ODA, at the level of the peripheral nervous system. Gene expression studies looking at expression levels for the recently characterized 9-ODA receptor,OR11 (Wanner et al., 2007 PNAS 104:36), in antennae shows receptor expression levels to be significantly higher in older mature drones and in young nurse bees as compared to immature drones and foragers, a pattern which coincides with periods of interaction and receptivity to the queen by the former groups. Though not significant, preliminary qualitative electrophysiological studies on workers show a consistent trend of increased sensitivity to the queen in the antennae of nurses vs. foragers, and a possible function of juvenile hormone in modulating olfactory sensitivity to 9-ODA. These findings may indicate a role for the peripheral nervous system in mediating, in part, the differential behavioral responses to the queen in workers and drones. 
10. Henderson, C.B. a, J.J. Bromenshenka, & D.L. Fischerb -- Clothianidin exposure levels from bee-collected pollen and nectar in seed-treated corn and canola plantings. aBee Alert Technology, Inc., 1620 Rogers Street, Suite 1, Missoula, MT 5902, bBayer Crop Science, LP, Research Triangle Park, NC 27709
    Field investigations of honey bee exposure to clothianidin from corn and canola grown from treated seed were conducted in 2010 and 2011. Fifty-three corn field sites, each consisting of 100+ acres of field corn grown from seeds treated clothianidin, were selected across three states, Illinois, Indiana and Nebraska. Pollen traps affixed to a single colony at each field were used to collect pollen gathered by foragers. A single bee-collected pollen sample was taken at each field, mid-tassel period in 2010.  Three samples spanning the tasseling period were collected in 2011. Pollen for comparison was collected directly from tassels in 2010. 
    Seed treatment was at 0.5 mg clothianidin/seed in IL and IN; 1.25 mg /seed in NE.  The former is the most common treatment rate in use in the US; the latter is maximum allowed by the label. Measured clothianidin residues in bee-collected pollen did not vary across the tasseling period, and the magnitude of residues were approximately proportional to the seed application rate. Consequently, values from 2010—treated at 0.5 mg/seed—were multiplied by 2.5 and pooled with those from 2011.  For the pooled data set (N=53 field sites), the mean clothianidin concentration was 1.2 ppb with 95% of residue concentrations below 2.8 ppb.  Thirty percent of bee-collected pollen samples were at or below LOD (0.44 ppb).
    Corn pollen in bee-collected pollen averaged just 19 ± 22 percent whereas corn fields comprised 72 ± 14 percent of habitat within one mile of the study colonies.  Clothianidin residue in tassel-collected pollen was higher than in bee-collected pollen averaging 4.4 ± 5.2 ppb with 95 percent below 11.9 ppb. There was a significant correlation between frequency of corn and clothianidin concentration in bee‑collected pollen, r=0.69, but there was no relationship between frequency of corn in bee-collected pollen and clothianidin concentration in tassel pollen, r=0.29. 
    Residues in canola were assessed in 2011 from thirty fields in southern Alberta, Canada. Bee-collected pollen averaged 1.7 ± 1 ppb and nectar averaged 0.8 ± 0.1 ppb clothianidin. Clothianidin concentration did not vary across the pollination period.  Bees made heavy use of canola pollen; it comprised 72 ± 26 percent of pollen samples.  Forty-four percent of bee-collected pollen samples were 100 percent canola. Unlike corn, there was no correlation between the concentration of clothianidin in bee-collected pollen and percentage canola pollen in the sample.
    We conclude from these findings that clothianidin residues in food items collected by bees are greater for bees placed at canola field sites than at corn field sites.  Even so, mean concentrations in pollen and nectar were 2 ppb or less and 95%tile levels were 4 ppb or less in these crop situations, levels that are not expected to pose a significant risk the honey bee colonies. Although residues in corn pollen sampled directly from tassels may reach 10 ppb or greater, preference by foraging bees for other kinds of pollen dilute their exposure to the lower levels we observed.  
11. Seccomb, R.A., C.B. Henderson,& J.J. Bromenshenk.  AUDIBLE CUES TO STRESS IN HONEY BEE COLONIES. Bee Alert Technology, Inc., 1620 Rogers Street Suite 1, Missoula, MT 59802
    Investigation into honey bee response to sublethal exposure to airborne toxicants showed that honey bee colonies produce unique and characteristic sound profiles when exposed to different toxicants. Furthermore sonograms from different classes of toxicant were distinct and could be statistically differentiated at near 100% correct classification.  Using these findings we explored whether other stressors of honey bee colonies induce similar identifiable sonographic profiles. We collected recordings of samples from free-flying colonies having verified conditions that included queenless and Africanized colonies as well as CCD, foul brood, small hive beetle, Nosema, and Varroa infections.  Each of these conditions produced similar, unique sonographic profiles. We have developed an artificial neural network algorithm that uses these sonographic profiles to quickly assess the presence of these conditions.  Using a microphone probe to make a 30 second recording, our instrument correctly identifies the presence of these conditions and the intensity of the infection with better than 85% reliability.  Prototypes of our device are being tested in the field to further refine and improve the instrument’s reliability in advance of its release for general use.
12.       Pernala, S.F., A. Ibrahima, S.E. Hooverb, R.W. Curriec, H.A. Higod, E. Huxtere, M.M. Guarnaf & L.J. Fosterf - PROTEOMIC MARKER-ASSISTED SELECTION IN HONEY BEES:  YEAR 2 UPDATE FROM THE BEEIPM PROJECT aAgriculture & Agri-Food Canada, Beaverlodge Research Farm, P.O. Box 29, Beaverlodge, AB Canada  T0H 0C0.   bAlberta Agriculture and Rural Development, Lethbridge Agriculture Centre, 100-5401 - 1 Ave South, Lethbridge, AB, Canada  T1J 4V6, cDepartment of Entomology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2, d1077 237A St. Langley, BC, Canada  V2Z 2Y2, eKettle Valley Queens, Grand Forks, BC, Canada  V0H 1H5, fUniversity of British Columbia, Department of Biochemistry & Molecular Biology and Centre of High-Throughput Biology, 2125 East Mall, Vancouver, BC, Canada  V6T 1Z4
    The Next Generation Integrated Pest Management Tools for Beekeeping (BeeIPM) Project aims to evaluate the efficacy of using proteomic marker-assisted selection for enhancing disease and Varroa destructor resistance in honey bee populations.  To evaluate the utility of this new tool for breeding, we embarked on a large-scale project.  In 2011, 622 colonies were phenotyped across four Canadian provinces for hygienic behaviour (HB). A portion of these colonies was then randomly selected to establish an unselected benchmark population (n=83) while an F0 population was established (n=110) from colonies most highly expressing HB.  We successively tested, selected and propagated two generations from our F0 during 2011 and 2012, in a parallel and direct comparison of proteomic-based marker-assisted selection (MAS) against traditional behaviorally-based phenotypic selection (FAS) on HB.  
    FAS-selected stock exhibited successive relative increases in hygienic behavior of 21.7 ± 2.4% and 45.7 ± 3.6% over benchmark populations in the F1 and F2 generations, respectively. Similar, though smaller, gains were observed for the MAS-selected stock where levels of HB increased 6.5 ± 2.8% and 29.2 ± 3.7 % over benchmark populations for the F1 and F2 generations. The F0 and F1 were also evaluated for Varroa Sensitive Hygiene (VSH) as described by Villa et al., 2009 (J. Apic. Res. 48: 162-167).  Though no significant differences were observed at one of two breeding sites in British Columbia, our Grand Forks F1 FAS selected stock showed reductions in mite infestations of 40.9 ± 6.0% while MAS stock showed reductions of 50.8 ± 5.0%.  F0 performance was documented at 25.8 ± 3.0%.
    Both FAS and MAS F1 selected stocks were also evaluated via whole-colony challenge experiments with American foulbrood disease (AFB) (Pernal et. al, 2008 J. Econ. Ent. 101:1095-1104). Evidence of differences in colony-level resistance to AFB were observed for several parameters, including the numbers of clinical symptoms in colonies over time and the number of P. larvae spores in workers collected from the brood nest. At the end of the twelve week evaluation period, 100% of colonies of the unselected New Zealand stock and 83% of Western Canadian benchmark exhibited symptoms of AFB.  In contrast, only 40% of MAS-selected and 15% of FAS-selected colonies exhibited symptoms.
    V. destructor resistance was evaluated by examining changes in total colony mite populations after a ten week period, in September 2012, and again in November.  Total mite levels in FAS and MAS selected colonies in the F1 did not significantly differ in November (means ranging from 2004 ± 234 to 2680 ± 1240) however, differences in mean mite abundance (mites per 100 bees) and adult bee population sizes were found in September and November. Mean abundance of V. destructor was lower in FAS and MAS colonies than in New Zealand colonies for both sampling periods, though similar to Western Canadian benchmark colonies. Bee populations in FAS colonies were larger than in benchmark colonies in both time periods, and FAS colonies were also larger than New Zealand stock by November.  
    Based on results to date, we conclude that selection on proteomic markers as well as traditional phenotype has enriched HB over two generations and has demonstrated initial proof of concept for proteomic selection in general. Detailed evaluations of the F3 will be made during the summer of 2013.

     Managed honey bee colonies are rented by fruit orchards to provide pollination services that improve fruit quality and yield. Placement of colonies in this agricultural setting increases the possibility of exposure to pyrethroids used for broad-spectrum pest control in orchards. Pyrethroids are highly toxic to bees (Smart and Stevenson, 1982 Bee World 63(4):150-152), and studies have correlated their use with decreases in honey bee foraging after application (Reviewed in Thompson, 2003 Ecotoxicology 12:317-330).       
    The goal of this study was to quantify sublethal behavioral effects associated with orchard-applied pyrethroid exposure in laboratory and semi-field situations. Quantification of sublethal effects may lead to more informed management decisions by growers and beekeepers. Development of video-tracking protocols may provide regulatory agencies with a risk assessment tool for measuring sublethal pesticide effects on pollinators.
    Following sublethal topical treatment of esfenvalerate, lambda-cyhalothrin, or permethrin, honey bee locomotion, time spent in a food zone and social interaction were quantified using video-tracking software, Ethovision XT® and methods from Teeters et al. (2012, Environ. Toxicol. Chem. 31:1349-1354). Separate analyses were performed on experimental colonies (A and B) and responses differed between colonies. Moderate (12.98 ng/bee) and high (25.96 ng/bee) sublethal doses of esfenvalerate significantly decreased total distance moved in colony A (moderate: p<0.0001; high: p<0.0001) and colony B (moderate: p=0.0195; high: p=0.0041). Social interaction time in colony A was significantly decreased at the highest dose of esfenvalerate (p<.0001). The highest dose of permethrin (52.29 ng/bee) significantly decreased both total distance moved and social interaction time in colony A (p<.0001; p<.0001). These results suggest that video-tracking can detect sublethal effects of esfenvalerate and permethrin on locomotion and social interaction at these doses.
    Repellencies of technical-grade esfenvalerate, lambda-cyhalothrin, and permethrin were measured at artificial feeders using methods adapted from Rieth (1986, Doctoral dissertation, University of Arizona). Control or treated filter paper was attached to polystyrene floats and placed in artificial feeders stocked with 20% sucrose solution and 30 ppm peppermint oil as an attractant. Contact pesticide exposure was simulated as foraging honey bees landed on the floats and the edge of the feeder in order to consume the sucrose syrup. Digital photos of the floats were taken every 10 minutes for 1.5 hours at each feeder. Using ImageJ software, digital images were examined to manually determine forager counts. Mean comparisons of forager counts over 10 time points were analyzed to assess repellency. Significantly fewer foragers were observed on permethrin-treated floats compared to control-treated floats at time points 3-10 (time point 3: p=0.0019; time point 4: p=0.0037; time point 5: p=0.0050; time point 6: p=0.0009; time point 7: p=0.0029; time point 8: p=0.0031; time point 9: p=0.0468; time point 10: p=0.0476)
14. Traver, B.E., N.G. Johnson, T.D. Anderson & R.D. Fell - EFFECTS OF PESTICIDE TREATMENTS ON PATHOGENS AND IMMUNITY IN HONEY BEE COLONIES - Virginia Tech University, Department of Entomology, Blacksburg, VA, 24061
   Honey bee colony losses are still occurring. While initially thought to be due to one factor, it is now probable that losses are influenced by multiple factors. The goal of this project is to examine the effects of pesticide treatments on pathogen levels and immunity of honey bees. Here we report the effect of 1) chlorothalonil, a commonly used fungicide, 2) fumagillin, the antibiotic used for Nosema control, and 3) tau-fluvalinate, an acaricide used for varroa mite control on Nosema ceranae and phenoloxidase levels. In the summer of 2012, colonies were established in apiaries that have not been treated with pesticides for five years. In the fall of 2012, colonies were either untreated (control) or treated with chlorothalonil (10 µg/L in sucrose solution), fumagillin (5 g/gallon in sucrose solution), or tau-fluvalinate (acaricide-impregnated strips; 10% w/w active ingredient). We collected samples of bees pre-treatment and 2 and 4 weeks post-treatment. For fall treatments, our results suggest that there was not a significant change in N. ceranae levels at any time point for the fumagillin-treated colonies compared to the untreated colonies. For chlorothalonil-treated colonies, there was a significant decrease in N. ceranae levels when comparing the pre-treatment and 4 weeks post-treatment (p = 0.03) time periods. Nosema ceranae levels also significantly decreased between the 2 and 4 weeks post-treatment (p < 0.01) time periods in these colonies. For tau-fluvalinate-treated colonies, there was a significant decrease in N. ceranae levels when comparing the pre-treatment and 4 weeks post-treatment (p < 0.01) time periods. Nosema ceranae levels also significantly decreased between the 2 and 4 weeks post-treatment (p < 0.01) time periods. Preliminary data for phenoloxidase activity suggest similar trends as N. ceranae levels for bees collected from the same pesticide-treated colonies.
15. Steinkampf, M.Pa, J. Hurstb & J. Tewc – EFFECTS OF OPTIMIZING HIVE SOLAR ABSORPTION ON HONEY BEE HEALTH AND PRODUCTIVITY. aSandhurst Bee Company, Mountain Brook, Alabama, bRockhurst Farm, Birmingham, Alabama, cAuburn University, Auburn, Alabama
    Ensuring that honey bee colonies maintain an optimal temperature remains one of the most debated and controversial topics of beekeeping. Although uncertainty about the benefit of conserving bee colony heat loss persists, wrapping hives in winter with tarpaper or insulation is still done by some beekeepers in colder climates. One potential reason for the inability to consistently document the benefit of hive insulation may be due to the fact that while insulation slows down the rate of energy exchange between the hive and the environment, it also limits penetration of heat generated via solar radiation. Preliminary experiments by our group indicated that honey bee hive temperature could be modulated using a hive coating that contained a thermochromic pigment, which appears black at low temperatures, facilitating solar absorption, but becomes white when temperature exceeds a threshold value.  The purpose of our study was to determine the effects of hive coatings that modulate solar radiation absorption on honey bee health and productivity.
     Hive boxes and covers were primed with white latex primer followed by two coats of TC coating (black/colorless, transition temperature 86F [31C], LCR Hallcrest Corporation, Streamwood, IL) and top-coated with four coats of transparent UV-protective spar varnish. Other hives coated with white (W) or black (B) latex paint to serve as controls.  In April 2011, we installed 13 three-pound honey bee packages from our local supplier after installing hives (5 TC, 5 W, 3 B) on single-beam platform scales.  Brood development was assessed after the colonies were established and in March of the following year, with honey harvest accomplished three months later.  During the study period, colonies were reestablished as needed so that all hives were occupied at the start of winter, and TC boxes were recoated in November to minimize the effect of pigment fading.  .
     Packages initially established colonies in all TC and black hive boxes, but in only 2 of 5 white hives (P=0.035). Total colony brood area 3.5 weeks after package introduction among established colonies was similar among the study groups (TC 561 in2, W 539 in2, B 674 in2; P=0.819).  Weight loss between November and the following January was also comparable (TC -5.2 lb, W -4.9 lb, B -1.9 lb; P=0.73), as was spring brood development (TC 604 in2, W 612 in2, B 701.8 in2; P=0.85) and honey yield (TC 40.5 lb, W 32.4 lb, B 21.3 lb; P=0.95).
     We conclude that hive coatings which increase the absorption of solar radiation in cool weather facilitate honey bee package installation, but otherwise they have no demonstrable effect on honey bee health or productivity.
16. Yang, W.C.a, H. Kuangb, J. Wanga, S.S. Wanga, Z.H Wua, X.Q. Miaoa, & Z. Y. Huangc COMPARATIVE SUCROSE SENSITIVITY IN APIS MELLIFERA AND A. CERANA FORAGERS.  aCollege of Bee Science, Fujian Agriculture and Forestry University, Fujian, Fuzhou 350002, China, bResearch Institute of Eastern Honeybee, Yunnan Agriculture University, Kunming, Yunnan 650201, China, cDepartment of Entomology, Michigan State University, East Lansing, MI 48824, USA
    Previous studies in the Western honey bee, Apis mellifera, have shown that pollen foragers have a lower sucrose threshold when tested using a proboscis extension response (PER) assay. Based on the biology of the Eastern honey bee, A. cerana, we hypothesized that A. cerana should have a lower threshold for sucrose. We compared the sucrose thresholds between pollen foragers and nonpollen foragers for A. cerana and A. mellifera in Fujian Province, China. Pollen foragers were more responsive to sucrose than nonpollen foragers in both species. Across the two species, A. mellifera was more sensitive than A. cerana in both types of foragers. In mixed species colonies where both species shared the same colony environment, A. mellifera also showed a higher PER score than A. cerana, so the higher sensitivity of A. mellifera was not due to a different colony environment. Based on these data, we predicted that nectar foragers in A. mellifera should bring in lower concentration nectar compared to that of A. cerana. We determined the nectar concentrations at each hour of seven-paired colonies of the two species of bees for seven days but found that the concentration of nectar foraged A. mellifera was not significantly higher than that of A. cerana.  There might be other mechanisms to enable A. cerana to perform well in areas with sparse nectar resources.
17. Zhou, S.J., X.J. Xu, X.J. Zhu, J.L. Gao, & B.F. Zhou * - GENETIC DIVERSITY OF APIS CERANA CERANA IN HAINAN BASED ON MITOCHONDRIAL DNA - Honey Bee Ecology Lab, College of Bee Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
    Population genetics of island species can provide us important information for their evolutionary history, genetic diversity and conversation. Hainan is a large island in south of China, with different environment and has been separated from the mainland for 10 million years. Apis cerana cerana is an important endemic species in Hainan. Even though previous studies have found 3 haplotypes by sequencing 9 honey bee workers in Hainan island(Jiang & ZHA., 2007 Sci. Agric. Sin. 40:7, Tan & Warrit, 2007 Apidologie 38:3), the sample size and number of collection sites were small. In this study, Apis c. cerana samples were collected from throughout Hainan island, and genetic diversity was determined by mitochondrial DNA fragments of tRNAleu-COII sequence.
    A total of  715 colonies were collected from Hainan Island, with 16 collection sites range from N 18°24.361′ to N 19°54.254′ and from E 108°50.359′ to E 110°52.689′. Workers were killed immediately by immersing them into absolute ethanol and kept at -80°C until processed in the laboratory. A single worker from each colony was examined by mitochondrial DNA. Mitochondrial analysis was carried out on a polymerase chain reaction produced from the intergenic tRNAleu-COII region (Garnery et al., 1993 Experientia 49:11), with the primers E2 located at the 5´-end of the tRNAleu gene, and H2 located at the 5´-end of the COII gene. An automated sequencer ABI3730 xl (Applied Biosystems, USA) was used to sequence the samples. Sequence data was aligned with the Clustal X1.83 program, and the haplotype diversity (Hd), average number of nucleotide differences (K) and nucleotide diversity (Pi ) were calculated by DnaSP5.00.07.
    A total of 48 haplotypes were found, 43 of which had not been previously published (Accession number: JQ323003-JQ3230045). There were 38 polymorphic sites, including 12 singleton variable sites, 23 parsimony informative sites, 1 insert site and 3 deletion sites. The average diversity of haplotypes was 0.742±0.017, the average nucleotide difference K and the nucleotide diversity of the honeybee samples Pi were 1.248 and 0.00354±0.00013 respectively, indicating the abundance of the haplotypes. The haplotype diversity (Hd) was unevenly distributed in all collections. The Wangning samples (WNLJ), from a southeast site in Hainan, were detected 3 haplotyes with the lowest haplotye diversity (1.171±0.066).Compared with the major haplotype (H02), the primary haplotype in Hainan island was H01, accounting for 46.15% of all samples in Hainan. But H02 was detected as the main haplotype in 3 collection sites (TCZJ, QZHM and QZJC), which were the primary places with introduced the mainland colonies. In addition, there were 74.27% honey bees in Hainan detected to have haplotypes which had the transition in 117th site (G-A), such a greater percentage was never detected in Chinese mainland.
    Mainland honeybee introduction and the rearing queen led to the obviously different genetic structure and lower abundance diversity in some places in Hainan, which was the reason of the unstable genetic structure in Hainan honeybee population. Results of this study will provide us the scientific basis for the conservation of Apis c. cerana in Hainan.
    The full study was recently published (in Chinese) in 2012, J. Fujian Agric. For. Univ. 41:2.
18. Matisoff, M. A. & T. C. Webster AN INEXPENSIVE TEST FOR NOSEMA IN HONEY BEES. College of Agriculture, Food Science and Sustainable Systems, Kentucky State University, Frankfort KY 40601, USA
    A simple and inexpensive test for the detection of Nosema spores in honey bee tissue is described. Currently, some beekeepers buy microscopes to observe spores in bee samples. But this is too expensive for many individuals. Apiary inspectors are available in some states to diagnose Nosema but this service may be time-consuming and it is not available in all states.
    The test depends on (1) the high density of Nosema spores compared to honey bee midgut tissue, (2) the ability of a stain, Calcofluor white, to bind to the chitin on the surface of the spore, and (3) the vivid fluorescence of the stain under ultraviolet illumination. Bee midguts are removed from the bees and mashed in water.  A drop of stain is added, the sample is mixed, and then it is passed through honey filter or similar material. The filtrate is allowed to settle in a small glass test tube for several hours. If the midguts are infected with Nosema, a pellet of spores will form at the bottom of the tube. The bottom of the tube is then illuminated with an ultraviolet-emitting diode powered by two flashlight batteries. Spores in the pellet will glow blue-white. The observations should be made in a very dark room.
    Observations of 100,000 spores are possible by this method. This is equivalent to a low infection in a single bee. A sample may consist of more than one bee, to improve the efficacy of the test. The entire system including stain costs less than five dollars.
    This test does not distinguish between Nosema ceranae and Nosema apis.  However, N. apis is now relatively rare in the U.S. The test is roughly quantitative if the observer compares the observed spore pellet to prepared reference pellets with known numbers of spores.  However this is not a research tool.
19. Webster, T. C., M. A. Matisoff  & C. Butler. EARLY DEVELOPMENT OF NOSEMA CERANAE IN HONEY BEE MIDGUT TISSUE. College of Agriculture, Food Science and Sustainable Systems, Kentucky State University, Frankfort KY 40601, USA 
     An understanding of the early stages of Nosema ceranae infection are important for two reasons. First, accurate observations of these early forms will allow tests of Nosema treatments to proceed quickly.  Similarly, efforts to breed bees resistant to Nosema will be facilitated if candidate bee stock may be inoculated and evaluated in short time periods. Second, the secretion and condition of the peritrophic matrix (PM) in the bee’s midgut is affected by N. ceranae infection.  PM condition may have other implications for bee colony health.
    We inoculated individual worker bees each with 50,000 N. ceranae spores, and later caged them collectively at 32 C with 50% sucrose as feed. Bees were removed every two days and examined for spores. N. ceranae infections are first visible by light microscopy, with the appearance of 1-2 million primary spores per bee 6 days post-inoculation (dpi). These spores appear oval, slightly tapered, and dark at one end using phase contrast optics. These spores peak at 8-12 dpi with 2-10 million spores per bee (mspb).  The mature form of N. ceranae, the environmental spores, are evident at 8 dpi (1-8 mspb). These are the spores that are shed in bee feces and are able to infect other bees. They steadily become more abundant, reaching 13-40 mspb by 22 dpi. From these observations we conclude that infections can be anticipated 6 dpi with primary spore counts.
    The PM is secreted by specific midgut epithelial cells. This structure is produced as a series of continuous concentric sheets in healthy bees. After the midgut is infected with N. ceranae it degrades to shreds. Other studies have shown important roles for the PM in insects: a barrier to ingested pathogens, a substrate for digestive enzymes, and protection against abrasion. We suggest these roles be considered in examinations of honey bee pathogens and nutrition.
20. Bernert, A., R. Sagili, & K. Johnson - EVALUATING FUNGICIDE SENSITIVITY OF THE HONEY BEE (APIS MELLIFERA L.) MICROBIOME. 4017 ALS Building, Oregon State University, Corvallis, OR 97331
    Recent honey bee colony declines have brought significant attention to the need of improving honey bee health and nutrition. The importance of the microbiome of the honey bee and hive has gained significant attention in the last few years (Mattila et al. 2012 PLOS One 7(4) e35954, Anderson et al. 2011 Insectes Sociaux 58:431-444). Microorganisms associated with A. mellifera L. have been shown to inhibit the growth of A. mellifera L. pathogens (Evans and Armstrong 2006 BMC Ecol 6(4). However almost all microflora molecular work has been devoted to bacterial communities while the metagenomics of fungi has not garnered significant attention.
    Some fungi have been found to be vital in the fermentation of pollen and making of  the beebread. Recent research findings have shown that fungicides negatively impact the growth of symbiotic beebread microbes (Yoder et al 2002 CRC Press. p 193-214). The microbes tested were morphologically identified to the taxonomic genus level thus excluding any ascomycete anamorphs. Also, fungal microflora may differ between geographical locations, and specific strains of the same genus or species may have significantly different resistance levels. Currently, there is no regulation on fungicide treatment at full bloom when honey bees are foraging. Nectar and pollen contaminated with fungicides are carried back to the hive. Fungicide residues have been found in the wax, beebread, and pollen (Mullin et al. 2010 PLoS One 5(3): e9754). In this study we evaluated interactions between honey bee gut microbes (fungi) and their sensitivity to common fungicides found in honey bee hive matrices.
    In this study we evaluated interactions between Apis mellifera L. gut microbes (fungi) and assessed their sensitivity to fungicides found in the hive matrices. Our results suggest that Chlorothalonil, Iprodione, and Boscalid inhibit growth of fungi associated with A. mellifera L. indicating the importance of further investigations into functional roles of these microbes and the effects of these fungicides in the hive. Fungicide concentrations used in this study were similar to residues found in wax and pollen. These concentrations inhibited growth of symbiotic fungi in vitro illustrating the need to know how they affect fungi in the hive and if those effects in turn would negatively impact colony health. Contrary to other studies, Chlorothalonil was detrimental to symbiotic fungal growth inhibiting nine of the ten isolates. Inconsistent trends of fungicide effects on fungal microbes, especially Mucor hiemalis, between this study and others highlights the need for further research into how exactly fungicides  effect different strains of symbiotic fungi.
21. Li, Z.G. a, Y.P. Chenb, S.W. Zhanga,c & S.K. Sua,d - EFFECTS OF IAPV ON FORAGING BEHAVIOR OF HONEY BEES (APIS MELLIFERA) - aCollege of Animal Sciences, Zhejiang University, Hangzhou 310058, China; bUSDA-ARS, Honey Bee Research Laboratory, Bldg. 306 BARC-E, Beltsville, MD 20705;cARC Centre of Excellence in Vision Science, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, Australia; dCollege of Bee Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
    Israeli acute paralysis virus (IAPV) has been detected in many parts of the world. Like other common honey bee viruses, IAPV plays a detrimental role on colony health, especially in combination with Varroa mite. Effects of IAPV on foraging behavior and homing ability of pollen foragers were therefore investigated in this study using radio frequency identification (RFID) system.
    Pollen foragers from colonies that weren’t detected with any of the seven common honey bee viruses were used in the study. Each of the twenty pollen foragers was injected artificially with 1 ul IAPV. The IAPV was diluted 1:200 in sterilized phosphate-buffered saline (PBS: 137 mM NaCl; 3 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO4, pH 7.2). Another group of twenty pollen foragers were injected with 1 ul PBS as a control. Each pollen forager was tagged with an RFID on the thorax and released 500 meters away from the hive. Data regarding foraging activity and homing ability of pollen foragers was collected by RFID readers placed in the hive entrance.
    We found that homing ability of foragers infected with IAPV was depressed significantly in comparison with foragers injected with PBS as a control. There was no significant differences (p >0.05) between the two groups in the number of foragers returning to the hive at days 0 and 1 post-injection. The number of foragers for PBS injected group returning to the hive at days 0 and 1 post-injection was around 16, which was nearly the same number for IAPV injected group. However, significant differences were found at 2 (p <0.01), 3 (p <0.05) and 4 (p <0.01) days post-injection between the two groups. Two days post-injection, there were 14 foragers injected with PBS returning to the hive compared with only 1 forager injected with IAPV returning to the hive. There were 10 PBS treated foragers left and returned to the hive 3 and 4 days after the injection, however, there were no IAPV treated foragers foraged 3 and 4 days after the injection. The homing experiment was repeated and carried out in three different colonies and similar results were obtained from three independent experiments. The data provide evidence that viral infection in the heads may make honey bees lose their way back to the hive.
22.  Wagonera, K., & O. Rueppellb. Contributions of Brood Communication and Mite-Exposure to Hygienic Behavior in Apis mellifera. aEnvironmental Health Science Program, University of North Carolina at Greensboro, Greensboro, N.C., USA, bDepartment of Biology, University of North Carolina at Greensboro, Greensboro, N.C., USA
     Honeybees (Apis mellifera) are important behavioral and molecular research models, and provide pollination services important to food security and maintenance of natural ecosystems worldwide.  However the last six decades have seen significant honeybee declines, and the rate of these declines has only increased in the United States over the past five years.  Honeybee declines are attributed largely to introduction and spread of novel parasites, specifically the recently introduced Varroa destructor mite, arguably the most important threat to apiculture today.  Additionally, pesticides, including many of the miticides used to control Varroa infestations, are known to have lethal and sublethal effects on honeybee queens, workers, and drones. Though Varroa resistance through hygienic behavior has already been successfully selected for in honeybees, inefficiency and non mite-specificity of these selection processes leaves much room for improvement.  Increased understanding and molecular markers of mechanisms underlying honeybee hygienic behavior are needed to improve existing selection processes. This research will study brood signals and the effects of mite pre-exposure on hygienic behavior to facilitate development of novel strategies for mite-specific selective breeding, which improve bee, human and environmental health by eliminating the need for chemical Varroa control. Experiments are being conducted to test the hypotheses that adult hygienic behavior is influenced by brood signals and by mite pre-exposure. Preliminary results support the hypothesis that adult hygienic behavior is influenced by brood signals, and suggest that these signals may be damage-dependent at least for certain honeybee genotypes. 
23.  Lichtenberg, E.M.a, J.S. Pettisb, M. Andreec, J. Stitzingera, R. Rosed & D. vanEngelsdorpa. -- COLONY HEALTH IMPLICATIONS OF POLLEN AND PESTICIDE DIVERSITY IN COLONIES RENTED FOR CROP POLLINATION – a College Park, Department of Entomology, 3136 Plant Sciences Building, College Park, MD 20742, b USDA-ARS Bee Research Laboratory, Bldg. 476 BARC-E, Beltsville, MD 20705, c University of California, Cooperative Extension Butte County, 2279-B Del Oro Ave., Oroville, CA 95965, d USDA-APHIS, 4700 River Rd., Riverdale, MD 20737
    Recent declines in honey bee populations and increasing reliance on insect pollination of crops raise concerns about pollinator shortages. Research suggests that pesticide exposure and pathogens interact to have strong negative effects on managed honey bee colonies. For example, exposure to sub-lethal doses of several insecticides increases bees’ susceptibility to or probability of death when infested with the gut parasite Nosema (Vidau et al., 2011 PLoS ONE 6:e21550; Pettis et al., 2012 Naturwissenschaften 99:153-158). Such findings are of great concern given the large numbers and high levels of pesticides found in honey bee colonies (Mullin et al., 2010 PLoS ONE 5:e9754). Thus it is crucial to determine how field-relevant combinations and loads of pesticides affect bee health.
    We collected pollen via pollen traps from hives rented for pollination of seven crops to ask two questions. 1) What pesticides enter the nest via pollen when bees are rented for pollination of various crops? 2) How do field-relevant pollen and pesticide blends affect honey bee susceptibility to Nosema infection? Focal crops were almond, apple, blueberry, cranberry, cucumber, pumpkin, and watermelon. First we identified pollens collected from each crop. Each pollen sample contained multiple pollen types. In most crops, no or little of the trapped pollen was from the target crop. The second part of this study analyzed pollen samples for pesticide presence and quantity. We detected 35 different pesticides in the trapped pollen. Fungicide loads were higher than loads of all other pesticide categories. Finally, we tested whether source and pesticides in pollen fed to bees affected those bees’ susceptibility to infection by the gut parasite Nosema ceranae. Several pesticides were associated with a higher risk of Nosema infection. In addition, probability of infection increased with the fungicide load in the pollen a bee was fed.
    Our results indicate that more attention must be paid to how bees are exposed to pesticides outside of the field in which they are placed, and to the sub-lethal effects of fungicides on honey bees.
24   Khongphinitbunjonga, K. L.I. de Guzmanb, M.R. Tarverb, T.E. Rindererb, Y.P. Chenc & P. Chantawannakula,d – PRELIMINARY RESULTS ON THE EVALUATION OF HONEY BEE STOCKS FOR SUSCEPTIBILITY TO DEFORMED WING VIRUS.  aDepartment of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand, bUSDA-ARS, Honey Bee Breeding, Genetics and Physiology Laboratory, 1157 Ben Hur Road, Baton Rouge, LA 70820, USA, cUSDA-ARS, Bee Research Laboratory, Beltsville, Maryland 20705, USA
dMaterials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
    We assessed the susceptibility of honey bee stocks to Deformed Wing Virus (DWV) infection. Three stocks (n = 4 colonies per stock) were evaluated: Italian (IHB), Pol-line (POL, hybrid Varroa Sensitive Hygienic bees) and Russian honey bees (RHB).
Each queen was caged to obtain uniformly-aged larvae. Rows of two-day old larvae were randomly fed 2µl of DWV lysate obtained by grinding 10 symptomatic bees in 10ml PBS. Several concentrations (DWV: PBS) were used: D1, D1:100, D1:1,000, D1:10,000, PBS alone and unfed larvae. Measurements were made of brood removal, DWV levels of newly emerged bees, weights at adult emergence and proportion of deformed bees. 
    Brood removal was universally about 30% (P = 0.99). For the levels of DWV, POL fed D1 had the highest while IHB and RHB had similarly low levels (P <0.0001, Figure 1). Newly emerged IHB fed D1 (115.2 ± 0.7 mg) and D1:10,000 (115.0 ± 0.6 mg) were the lightest (P <0.0001).  Emergence weights of POL (117.6 ± 0.8 mg) and RHB (113.0 ± 0.6 mg) were higher and comparable. Larvae fed D1 (5.84 ± 1.64%) showed the highest proportion of deformed bees (P = 0.005).  D1:10,000 (1.26 ± 0.52%) and control (0.90 ± 0.46%) groups had the lowest while D1:100 (3.43 ± 0.66%), D1:1,000 (2.55 ± 0.99%) and PBS (1.70 ± 0.63%) were intermediate.  The proportion of deformed bees differed (P = 0.049): IHB (3.57 ± 0.68%) ≥ POL (2.39 ± 0.69%) ≥ RHB (1.40 ± 0.49%). Based on the low DWV levels and proportions of deformed bees, RHB may have some tolerance to DWV infection.
Figure 1 – Levels of DWV of three different honey bee
stocks fed different concentrations of DWV.

25.  de Guzman, L.I., T.E. Rinderer & A.M. Frake – DOES THE REMOVAL OF MITE-INFESTED BROOD FACILITATE GROOMING? USDA-ARS, Honey Bee Breeding, Genetics and Physiology Laboratory, 1157 Ben Hur Road, Baton Rouge, LA70820
 The relationship between the removal of mite-infested brood and mite drop was compared using Russian (RHB, n = 9) and Italian (IHB, n = 9) honey bee colonies. A cloake board was used to isolate test brood frame on the top hive body and the metal sheet served as a varroa trap. Inoculum mites were collected from newly sealed larvae and each was marked using correction fluid (Kirrane et al., 2012 J. Apic. Res. 51: 212-213). Brood cells randomly received one of the following groups: 1) brood inoculated with one female varroa, 2) brood with capping opened and closed without mite inoculation (o/c), and 3) undisturbed brood cells as control. Brood removal and mite drop were determined every day for eight days.
    Both stocks removed more mite-inoculated brood than o/c or control groups (P <0.0001). RHB (87.9 ± 2.0%) significantly removed more inoculated brood than IHB (61.9 ± 7.3%) (P = 0.0001). Increased removal of frozen brood by RHB has also been demonstrated (de Guzman et al., 2002 Am. Bee J. 141: 58-60). Although both stocks removed brood every day, brood removal peaked during the first four days for IHB and during the first two days for RHB colonies. Overall, the RHB (2.5 ± 0.1 days) removed brood faster than the IHB (3.0 ± 0.1 days) colonies (P = 0.014).
    Fallen marked mites were collected from traps every day with peaks observed during the first three days coinciding with the peak of brood removal. Overall, about 35% of the introduced mites dropped from the RHB compared to 24% for IHB. A similar observation was reported by Rinderer et al. (2001 Apidologie 32: 381-394). Regardless of stock, the number of dropped mites increased with an increase in brood removal (r = 0.089, P = 0.0001) (Figure 2). Brood removal may be one of the major causes of high mite drop in honey bee colonies.

Figure 2 – Correlation between the removal of brood inoculated
with varroa mites and number of marked mites that dropped on traps.

26.  Holloway, Ba., J.W. Harrisb,  J.Villaa,  R. Dankaa – FINE MAPPING FOR SNP MARKERS ASSOCIATED WITH VSH BEHAVIOR –  aUSDA-ARS, Honey Bee Breeding, Genetics and Physiology Lab, 1157 Ben Hur Road, Baton Rouge, Louisiana 70820. b Mississippi State University, Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology.
     Varroa Sensitive Hygiene (VSH) is a trait that effectively reduces varroa mite populations by removal of brood cells that contain primarily reproductive mites.  Breeding for VSH has proven to be a successful control of mite populations in both pure VSH colonies as well as in out-crossed populations.  Selection of queens that carry the hygienic trait requires colony-based phenotyping beyond the reach for most bee breeders.   Marker assisted selection of VSH queens could substantially reduce the labor requirements of that for classical selection while increasing the overall VSH trait in the breeding stock. 
     Quantitative Trait Loci (QTL) mapping (Tsuruda et al.,2012 PLoS ONE 7(11)) identified a genomic interval that associates with the VSH trait.  The QTL contains several candidate genes that could be involved in the VSH trait in bees based on known functions in other model systems.  We performed fine scale mapping to refine the QTL interval and identify additional molecular markers that could be used for marker assisted selection.  Several markers located within two particularly interesting candidate genes associate strongly with the phenotype.  mRNA sequencing suggests that a splice variant of one of the genes may result in the VSH trait.  The strongest associating marker was used to genotype 28 colonies of either Italian or VSH stock in a single-blind study.  Predictions for colony phenotype based on marker genotype proved to be accurate for 89% of the colonies when a strict set of prediction criteria were followed.  This validation of a strong molecular marker associating with the VSH trait suggests that marker assisted selection may be highly effective for increasing VSH in breeding stocks at reduced costs and effort.
27. Dahlgren, L.a, E. Siegfrieda, R.M. Johnsonb, B.D. Siegfrieda & M.E. Ellisa ‑ HONEY BEE CASTE-SPECIFIC ACETYLCHOLINESTERASE INHIBITION: INSIGHTS INTO COUMAPHOS TOLERANCE. –  a University of Nebraska,103 Entomology Hall, Lincoln, NE, b The Ohio State University, Thorne Hall, 1680 Madison Ave., Wooster, OH. 
    Acetylcholinesterase functions in the central nervous system to regulate nerve impulse transmission and is the target for organophosphate insecticides such as coumaphos.  Queens are 11 fold more tolerant of coumaphos than workers (Dahlgren et al. 2012 J. Econ. Entomol. 106:1895-1902).  We evaluated whether differences in target site sensitivity could explain differences in worker and queen responses to coumaphos.  Acetylcholinesterase activity can be measured with a simple spectrophotometric activity assay known as Ellman’s assay.  We compared honey bee worker and queen acetylcholinesterase inhibition by two organophosphate metabolites: coumaphos oxon and chlorpyrifos oxon.  We also examined the response of a susceptible house fly (Musca domestica L.) population to establish whether honey bees respond differently than other insects.  Acetylcholinesterase preparations were collected by homogenizing the heads of both honey bees and houseflies known to contain high concentrations of acetylcholinesterase.  Inhibitors were diluted in ethanol and incubated with enzyme at 37°C for 5 time periods.  Absorbance was measured at 21 second intervals for 5 minutes (at 412 nm), converted, and expressed as nmoles substrate hydrolyzed per minute per milligram protein (Siegfried and Scott, 1990 Pestic. Biocem. Physiol. 38:122-129).  Results to date indicate that there is no apparent difference between queen, worker, and fly acetylcholinesterase inhibition for either coumaphos oxon or chlorpyrifos oxon.  This indicates that queen tolerance of coumaphos is not due to target site insensitivity.  Continued study of the mechanism of coumaphos tolerance in honey bees may lead to the design of acaricides with minimal adverse effects on colony health.
28.  Caron, D.M.a,  R. Sagilia & M. Cooperb – 2012 Survey OF PNW Pollination rentals –  a Affiliate faculty and Assistant Professor respectively, Department of Horticulture, Oregon State University, Corvallis, OR-97331-7304; bBureau chief, Division of Plant Industries, ID Dept of Agriculture, Boise, ID 83701
    We have a tradition of surveying Pacific Northwest (PNW) beekeepers (OR, ID and WA) for both bee losses and pollination economics. Approximately 200 single page surveys were sent via US mail (stamped return envelope) requesting information on number of colonies rented by individual crop and rental price received during 2012. This was the 27th year of this survey; results of the first 25 years were annually presented by Burgett (2000-11. various issues USDA Honey Market News & Am Bee J 150(1):35-40) and by present authors for 2011 (Caron, et al 2012. Am Bee J 152(5):503-506). This report is preliminary results of 2012 survey.
For 2012, we have tallied responses from 44 individuals managing  an estimated 48% of the colonies in the 3-state region. The weighted average rental (total of slightly almost 200,000 rentals with value over $17 million) was down 9% at $84.50.
    For comparison our 2011 survey included 63 beekeeper responses representing 70% of estimated beekeepers with 5 or more honey producing colonies (USDA, NASS). Weighted average pollination fee was $90.62 for almost 254,000 rentals of total value of just under $23 million. For both years there were 4.2 average rentals reported by the beekeepers. This included almond pollination  rental in California for all commercial and the majority of 4 semi-commercial individuals plus 12 other crops.
    The weighted pollination fee for almonds and major PNW rental crops for past dozen years is shown in Figure 3 below.

29.  Tsuruda, J.M.a, Subramanyam, S. a, Arechavaleta-Velasco, M.E.b, Alcala-Escamilla, K.I. b, Robles-Rios, C. b, Williams, C.E. a, & G.J. Hunt a – TESTING GENE EXPRESSION LEVELS OF NEUREXIN I IN BEES EXHIBITING HIGH AND LOW GROOMING BEHAVIOR – aPurdue University, 901 West State St., West Lafayette, IN, bInstituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Ajuchitlan, Qro. Mexico
    Grooming is an important behavioral trait of honey bees that reduces Varroa mite populations. Adult bees attempt to dislodge mites from their bodies by swiping movements of their legs and may damage the mite by biting. Selective breeding for this trait would be useful for beekeepers and the identification of the genes involved could allow for marker-assisted selection and facilitate the breeding of mite resistant bees.
    We previously conducted a study to look for associations between phenotype (grooming behavior) and genotype by mapping quantitative trait loci (QTL). We used a behavioral assay that measured the time for a bee to respond to a mite placed on her thorax. One QTL was identified that contained 27 candidate genes, three of which are associated with neurological processes that could play a role in grooming. Neurexin 1 is a particularly interesting candidate as it is involved in autism spectrum disorder and schizophrenia in humans, which can involve repetitive and agitated movements and altered sensory responsiveness, and grooming behavior in mice. This is a relatively large gene with 28 exons and two major forms, with at least 12 different isoforms due to alternative splicing. We hypothesize that these major forms, or relative amounts of these forms, affect the level of grooming behavior in worker honey bees and use qRT-PCR to measure the expression of the two major forms of neurexin I.
    We performed our behavioral grooming assay and measured response time and grooming intensity. The preliminary data presented here is from six ‘good’ groomers and six ‘poor’ groomers, selected based on their response time and grooming intensity. Primers were designed to target each of the major forms of neurexin I. qRT-PCR was preformed in triplicate with AmRPL8 as a housekeeping gene and data was analyzed using the ΔΔCt method.
    We found that the two major isoforms show similar relative expression patterns. Within a form, we did not see a difference between ‘good’ and ‘poor’ groomers but a trend was observed between gene expression and response time. This data is preliminary and we will sample more bees for neurexin-I expression to look for stronger support of the involvement of neurexin I in honey bee mite grooming behavior. Future directions include RNAi to validate gene function and characterization of neurexin I in A. cerana. Ultimately, we aim to use this gene to develop a breeding program for behavioral resistance to Varroa utilizing marker assisted selection.
30.  Hunt, G. J.a, S.D. Kocherb, J.M. Tsurudaa, C. Emorea, M.E. Arechavaleta-Velascoc, C. Robles-Riosc, C. Grozingerd, D.C. Queller5 & J. Strassmane  PARENT-OF-ORIGIN EFFECTS ON GENE EXPRESSION IN LARVAL AND ADULT HONEY BEES.   aPurdue University, 901 West State St., West Lafayette IN,  bHarvard University, 26 Oxford St., Cambridge MA,  cInstituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias (INIFAP), Ajuchitlan, Qro. Mexico.,  dPennsylvania State University, 501 ASI Building, University Park PA, eWashington University, One Brookings Drive, St. Louis MO
    We investigated whether parent-of-origin (PoO) effects influenced gene expression by sequencing gene transcripts (cDNA) from reciprocal hybrid workers derived from crosses between one European and one Africanized colony.  Sequencing was done on the Illumina HiSeq machine in the Purdue Core Genomics Facility. We identified heterozygous single-nucleotide polymorphisms (SNPs) that were identical in both families and then determined whether the maternal allele or paternal allele was over-expressed by comparing the number of read counts. We also sequenced genomic DNA from the parents to determine the parental source for each allele and also genomic DNA from one of the families to eliminate SNP data that did not agree with parental genotypes. Two pools of individuals were sequenced for two life stages in each family (4 pools of 1st instar larvae and 4 pools of adults). We pooled the maternal and paternal alleles for transcripts that had more than one SNP and performed a general linear interactive analysis of variance for the counts of number of reads per allele after square-root transformation. We also required that transcripts that were called parentally biased show the same overall bias (maternal or paternal) in both families.  About 2,500 hundred gene transcripts could be analyzed in larvae and 1,500 in adults.
    The results indicate an overwhelming tendency towards maternally biased transcription. Approximately 150 transcripts showed a significant parental bias in larvae and 100 transcripts showed a PoO effect in the adult workers. In both life stages 91% of the transcripts showing a parental effect were biased towards the maternal allele.  In contrast, about 60% of mammalian genes that are known to be parentally biased show a maternal effect. A paternal bias could potentially give an advantage to one subfamily over others. A maternal bias may in general be better for the whole colony fitness. There appeared to be many maternally biased genes that are known to be involved in oxidative metabolism, energy homeostasis, mitochondrial function, insulin/insulin-like signaling, and lipid metabolism. This suggests that genes that work together in critical gene networks are more likely to be expressed from the queen’s allele. These results could be subject to bioinformatic artifacts so further study is needed to validate these effects.

31. Eischen. F.A., R. H. Graham, & Rivera. WHY DO THE BEST COLONIES HAVE A HIGH RATIO OF SEALED TO TOTAL BROOD? Carl Hayden Bee Research Center, Tucson, AZ  85719
      We examined the survival and developmental times of a cohort of eggs laid in colonies (n = 5) fed either a) almond pollen (fresh frozen Feb 2011), b) 10% almond pollen + Bee Pro, c) 10% Chinese pollen + Bee Pro, or d) Bee Pro. Immediately prior to this study, colonies were fed these diets for 82 days (20 Oct 2011- 10 Jan 2012).  Colonies (n = 25) during this prior study were able to perform continuous brood rearing on all diets. Differences in performance leading up to the current study suggested that immature survival and developmental times were contributing factors to smaller colony size. In this study, those fed the almond pollen diet had the highest immature survival (97.5% versus 29.7% for Bee Pro). Developmental times were quickest for the two best performing groups. On day 20, 74.2% of sealed brood had emerged for almond-fed colonies, while only 10.2% of sealed brood reared on 10% Chinese pollen + Bee Pro had emerged.  Further, almond-fed colonies had significantly heavier newly emerged workers; and their soluble protein levels were higher.   
     The immature developmental survival rates provided insight into why the improved performance by almond-fed colonies was coincident with a high percentage of sealed brood. In several prior studies involving large numbers of commercial colonies, we have consistently observed that the best performers have a high ratio of sealed to total brood. The data in this study indicates it was primarily due to significantly higher open brood survival combined with shorter time spent in the open brood stage. The poorest performing group (Bee Pro) lost on average 76.1% of their open brood. A slightly lengthened sealed (pupal stage) may also have been a factor for the better performing colonies. Colonies with low ratios of sealed brood exhibit high immature death. We suspect strongly, that dead larvae are removed and new eggs laid.  Thus the low ratio of sealed to total brood. With a modest amount of practice, beekeepers can recognize low/normal/high sealed brood ratio colonies. This is a good indicator of colony nutrition and performance during winter feeding.   

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