Progress 03/01/11 to 02/29/16
Outputs
Target Audience:Target audiences include the scientific community (entomologists, toxicologists, geneticists, andphytochemists) and the apiculture community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two students worked on this project in its final year; both gained experience designing and conducting behavioral and physiological experiments with honey bees. Beyond these skill sets, Ling-Hsiu Liao increased her mastery of molecular analytical techniques, including qRT-PCR. Catherine Dana expanded her familiarity with image processing software (to analyze video recordings of honey bee feeding behavior), as well as statistical analytical techniques, and will deposit her MS thesis this summer. Both Liao and Dana will be submitting manuscripts for publication acknowledging this grant. How have the results been disseminated to communities of interest?In the final year of the project, dissemination of findings to the scientific community included: "The pollinator crisis: Are America's bees, flies and butterflies on their last six legs?" and "Can science save the honey bee?" Quad Cities Pollinator Conference, U.S. Forest Service, Rock Island, IL, June 10, 2015; 2015. "The A-Bee-Cs of pollinator K-3 education" Beginning STEM teachers conference, Champaign, IL, July 29, 2015. "Honey bees as phytochemists" Annual meeting Phytochemical Society of North America, Urbana, IL, August 8, 2015. "Pollinators and CYP blooms," Asia-Pacific Association of Chemical Ecologists, Anaheim, CA, September 28, 2015; Plenary, Sigma Xi Annual meeting, John P. McGovern Award Lecture, Kansas City, MO, October 24, 2015. "Apis mellifera: Can a 6-million-year-old genome survive the Anthropocene?" Keynote, Entomological Society of Canada annual meeting, Montreal, Quebec, November 11, 2015. Member symposium "CCD: Eight years later," Entomological Society of America annual meeting, Minneapolis, MN, November 15, 2015; White House Executive Office Transportation Leaders Pollinator Summit, Washington DC, December 3, 2015. Dissemination of findings to the general public included: "Declining bee populations" Lauren Williamson, Chicago Magazine,June 30, 2015. "Beespotter" Interview with Anthony Watt, Moline Dispatch and Rock Island Argus. "How I became a scientist" Pollen Power program for middle school girls, Urbana, IL July7, 2015; Interview about bumble bees with Malcolm Ritter, Associated Press, July 7. "Saturday Night Hive: Can science save the honey bee?" Saturday Night Live at Clark-Lindsey Retirement Village, Urbana, IL, July 11, 2015. "Honey bee update: The hive as Whoville" Pollination Fascination, UI Pollinatarium; Science Advances--Ashley Taylor, The Scientist; Gwen Pearson, Wired; Tim Wogan, Chemistry World; Sarah Everts--Chem & Eng News; Science Soiree South Pasadena, CA, September 24, 2015; Integrative Illini, October 21, 2015. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the final year of the project, we carried out RNASeq analysis of gene expression in newly hatched larvae reared for three days on diets with and without quercetin, a ubiquitous flavonol found in honey, beebread and propolis, and found that, in addition to upregulating multiple detoxifying P450 genes, quercetin is a strong negative transcriptional regulator of mitochondria-related nuclear genes and thus can interfere with energy production. In silico high-throughput docking of 121 pesticides and metabolites found in North American bees and hives into the active pocket of CYP9Q1, one of several enzymes that metabolize quercetin, identified 68 compounds that could be docked, including 6 triazole fungicides, which are known P450 inhibitors. In vitro experiments with intact midguts verified that the triazole myclobutanil inhibits quercetin-metabolizing P450s in adult worker bees; bees that consume quercetin in the presence of myclobutanil produce lower levels of ATP, and thus energy for flight muscles, in their thorax. Although fungicides may not be overtly toxic to bees, they clearly influence their health by rendering them less efficient at metabolizing quercetin, allowing it to accumulate in the thorax and compromise mitochondrial regeneration and ATP production. The widespread use of triazole fungicides in combination with insecticides, including neonicotinoids, for systemic seed treatment essentially guarantees that bees will encounter and ingest pesticides in combinations likely to reduce their ability to detoxify quercetin and thus to extract the energy they need from the food they consume. In addition to examining impacts of quercetin on gene expression, we undertook an experiment to elucidate functions of CYP4G11 in the honeybee genome by comparing its expression relative to worker age and task to expression of CYP9Qs known to metabolize xenobiotics. That CYP4G11 is highly expressed in forager antennae and legs, with highest expression in prothoracic and mesothoracic legs, is consistent with chemosensory perception, whereas weak expression of CYP4G11 in nurses suggests it may primarily process exogenous rather than endogenous chemical signals. In contrast, and consistent with xenobiotic detoxification, the three CYP9Q transcripts are almost undetectable in newly enclosed workers and highest in foragers, with highest expression in metathoracic legs that closely contact pollen phytochemicals. These CYP4G11 expression patterns suggest a role in processing environmental signals, particularly those associated with food.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Mao, W., M.A. Schuler and M.R. Berenbaum, 2015. Task-related differential expression of two cytochrome P450 genes in honeybee appendages. Insect Molecular Biology 24: 582-588.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Mao, W., M.A. Schuler and M.R. Berenbaum. 2015. A dietary phytochemical alters caste determination gene expression in honeybees. Science Advances 1(7): e1500795.
DOI: 10.1126/sciadv.1500795.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2016
Citation:
Mao, W. and M.R.Berenbaum. 2015. Fungicide compromises energy production by honeybees ingesting floral flavonoid. Under Review.
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Progress 02/28/14 to 02/27/15
Outputs
Target Audience: Target audiences include the scientific community (entomologists, toxicologists, geneticists, phytochemists) and the apiculture community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three students (Ling-Hsiu Liao, Catherine Dana, and Michael Wong) have gained experience designing and conducting behavioral and physiological experiments with honey bees. Liao's work on caste and age-related variation in esterase activity is the basis of a publication in preparation and will be included in her doctoral thesis; Liao and Dana collaborated on a behavioral experiment testing whether free-flying bees display aversive responses to phytochemicals and pesticides and this work is being prepared for publication and will be included in Dana's master's thesis. How have the results been disseminated to communities of interest? In 2014-2015, the following activites involved dissemination to the scientific community: (1) The 45th annual meeting of the Australian Entomological Society, September 29, 2014; (2) Examining the Environmental Effects of Practices for Controlling Agricultural Pests (you can't win), Workshop on Comparing the Environmental Effects of Pest Management Practices Across Cropping Systems, Board on Agriculture and Natural Resources, National Academy of Sciences, Washington DC , March 5, 2015 (via Webex); (3) 30th Anniversary Ecology and Evolution Department National Academy Lecturer, University of Connecticut, Storrs, March 24, 2015; and (4) 10th Annual Rieveschl Lecture, Department of Biology, University of Cincinnati, March 25, 26 (two lectures). In 2014-2015, the following activities involved dissemination to beekeepers and the general public: (1) Aninterview onMarch 17, 2015 forBBC World Service. What do you plan to do during the next reporting period to accomplish the goals? Planned are presentations at the: (1) Entomological Society of Washington, June 4, 2015; (2)Quad City Pollinator Conference (USGS), June 10, 2015; and (3) University of IllinoisPollinatarium, Urbana IL, June 21, 2015.
Impacts
What was accomplished under these goals?
For Objective 1,we foundp-coumaric acid, pinocembrin, and pinobanksin 5-methyl-ether induce multiple detoxification genes in adults and larvae. Consuming sucrose with p-coumaric acid enhanced coumaphos detoxification by 60%, demonstrating its functional role (Mao et al. 2013). Regarding Objective 2 and Objective 3, we showed cytochrome P450s CYP9Q1-CYP9Q3 metabolize tau-fluvalinate and coumaphos; these compounds compete for access to the catalytic pocket, explaining their synergistic toxicity. CYP9Q2 and CYP9Q3 transcripts are upregulated by honey and heterologous expression studies confirmed CYP9Q3 metabolizes the flavonoid quercetin. qRT-PCR assays demonstrated fluvalinate enhances CYP9Q3 transcripts, whereas bifenthrin enhances CYP9Q1-Q2 and represses CYP9Q3 (Mao et al. 2011; Johnson et al. 2012). As for Objective 4, RNA-seq showed p-coumaric acid upregulates detoxification genes in larval workers and alters expression of caste-determining genes, upregulating DNA methyltransferase-3 and down-regulating royalactin and insulin-like peptide-2 genes. That larvae fed royal jelly with p-coumaric acid become adults with reduced ovaries suggests royal jelly both enriches the diet of queen-destined larvae and protect them from inhibitory phytochemicals (Mao et al., in revision). Also, at low concentrations quercetin upregulates detoxification genes but at high concentrations down-regulates 18 genes encoding the large and small units of mitochondrial ribosomal proteins and mitochondrial transcription factor B2. Consequently, assembly of Complex I, III, IV and V of oxidative phosphorylation and hence energy production are likely compromised.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Berenbaum, M.R. Does the honey bee 'risk cup' runneth over? Estimating aggregate exposures for assessing pesticide risks to honey bees in agroecosystems. J. Ag. Food Chem. 2015 May 11. [Epub ahead of print].
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Berenbaum, M.R. and R.M. Johnson. 2015. Xenobiotic detoxification in the western honey bee Apis mellifera. Curr. Opinion Insect Science. http://www.sciencedirect.com/science/journal/aip/22145745.
- Type:
Conference Papers and Presentations
Status:
Awaiting Publication
Year Published:
2015
Citation:
Berenbaum, M.R. 2015. Bees in crisis: Colony collapse, honey laundering, and other problems bee-setting American apiculture. Proc. Am. Phil Soc. [In Press].
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Berenbaum, M.R. 2015. Xenobiotic detoxification pathways in the honeybee Apis mellifera. Current Opinion in Insect Science: 05/2015; 10. DOI: 10.1016/j.cois.2015.03.005.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Liao, L.-H. and M.R. Berenbaum. 2015. Variation in ester metabolism with caste and temporal polyethism in Apis mellifera. Submitted to Apidologie.
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Progress 02/28/13 to 02/27/14
Outputs
Target Audience: The target audience for this research includes academic researchers in the fields of entomology and toxicology as well as beekeepers and others in the agricultural sector interested in managed pollination. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has contributed to the training of one postdoctoral student (Wenfu Mao), two graduate students (Catherine Dana, Ling-Hsiu Liao) and several undergraduates who assisted in gathering and compiling data. How have the results been disseminated to communities of interest? Scientific findings were disseminated in refereed journals aimed at the research community. In terms of public audiences, activities included: June 12, 2013, interview on honey chemistry for digital magazine The Column, LCGC; June 14, 2013, interview for digital magazine Vetstreet.com on bee health; “Applied Bee-nomics,” Congressional Hill Lunch-n-Learn seminar, June 16, 2013; Stakeholder meeting, “Open dialogue—research priorities, translating research from hive to honey and other products”, American Farm Bureau, CropLife America, June 16, 2013; Kathleen Dunn call-in show, “Honeybee health,” Wisconsin Public Radio, June 19, 2013; "Pollination fascination—pollinator genomics”, UI Pollinatarium, June 30, 2013; New technologies for U.S. apiculture, Suzanne Zionts and Stephanie Becker, Al Jazeera America, August 5, 2013; Alexandra Sifferlin, Time.com, entomophagy and food security query, August 20, 2013; Neonicotinoids and pest control operators, Ann Nagro, Pest Control Technology magazine, August 26, 2013; Interview and podcast with Adrian Smith, aodpod.com, Origins, September 4, 2013; Interview with Seth Shostak, “Disappearing bees,” Big Picture Science radio and podcast, September 4, 2013; Interview, “Is there a bee crisis?” Richard Wills, Pittsburg Tribune-Review, September 13, 2013; George S. Saul Public Lecture, Middlebury College, Middlebury VT, September 19, 2013;“How the honey bee genome project is revolutionizing pollinator research” AgMasters Conference, Champaign, IL December 2, 2013; Interview with Joel Brownstein, LiveScience, on tobacco hornworm defense; interview with Susan Milius, Science News, on tobacco hornworm defense, December 30, 2013; Interview with Paul Wood, Champaign-Urbana News Gazette, powder-post beetles in Spurlock, “Exhibiting an intolerance for pests,” January 18, 2014; Interview with Voice of Russia, Bees and Pesticides, London, UK, January 24, 2014; Interview with Seth Borenstein, Associated Press, “Zom-bees”, January 29, 2014; Pesticides, WILL AM Focus 580, February 18, 2014; Interview with Seth Borenstein, Associated Press, disease associations between honey bees and bumble bees, February 19, 2014. George S. Saul Lecture, Department of Biology, Middlebury College, Middlebury VT, September 20, 2013; “Predicting the future of entomology—lessons from the past,” First Annual Richards-Hodson Lecture, Department of Entomology, University of Minnesota, St. Paul, MN, October 24, 2013; “Bees and pesticides and gypsy moths,” Annual Gypsy Moth Review, Evanston, IL, November 5, 2013; Entomological Society of America Annual Meeting, Austin, TX: “Does the honey bee risk cup runneth over?” P-IE Section Symposium: Beyond the LC50: Advancements in Toxicological Research on Pollinators, November 10, 2013; “Impacts of landscape-scale conversion to bioenergy crops on microlepidopteran diversity: Biofuels and LBMs”, P-IE Section Symposium: Biofuel Cropping Systems: Connecting Beneficial Arthropods, Ecosystem Services, and Landscape Effects, November 12, 2013; “Farming out pharmacology: Symbiont detoxification of phytochemicals",P-IE Section Symposium: The Effect of Microbes on Insect-Plant Interactions, November 12, 2013; You are what you eat: honey and the honey bee", P-IE Section Symposium: Connecting Research, Outreach and Regulatory efforts to Protect Honey Bee Health, November 12, 2013. “Honeybee cytochrome P450s: how a 6-million-year-old genome copes with pesticide-intensive modern agriculture” at “Impact of Pesticides on Bee Health” meeting, Society of Experimental Biology/Biochemical Society/British Ecological Society, London, UK, January 22-24, 2014; Friday Seminar Programme, John Innes Plant Research Center, Norwich, UK, March 24, 2014; Rochester Community and Technical College Environmental Science Program, Bees in Crisis: Colony Collapse, Honey Laundering and Other Problems Bee-Setting American Apiculture, Rochester, MN, April 8, 2014; Storer Lectures, Department of Entomology, University of California at Davis, Davis, CA, May 20-21, 2014; Back Yard Beekeepers, Weston CT May 27, 2014. What do you plan to do during the next reporting period to accomplish the goals? Upcoming presentations are scheduled with beekeeping groups in Maryland and Illinois during summer 2014 and scientific presentations will be made at the November 2014 Entomological Society of America meeting.
Impacts
What was accomplished under these goals?
Our specific objectives and our progress to date are: [1] To identify inducers and inhibitors of cytochrome P450 detoxification gene transcription in naturally occurring (nectar, pollen) and processed (honey, beebread) components of the honey bee diet. With respect to this objective, we identified four constituents of honey (p-coumaric acid, pinocembrin, pinobanksin and pinobanksin 5-methyl ether) that upregulate detoxification and immunity genes in both larval and adult bees; [2] To determine how in-hive and agricultural pesticides induce or inhibit P450 detoxification. With respect to this objective we determined that CYP9Q enzymes are involved in detoxification of acaricides and that tau-fluvalinate and coumaphos compete for access to the catalytic site and synergize each other; [3] To identify specific P450 enzymes that metabolize pesticides and phytochemicals. With respect to this objective we identified CYP6AS genes as encoding enzymes that metabolize flavonoids in honey and beebread/pollen and CYP9Q genes that encode enzymes that metabolize naturally occurring flavonoids as well as in-hive and agricultural pesticides; and [4] To test whether diet (honey, sucrose, high fructose corn syrup) influences transcription of P450 genes and toxin tolerance. With respect to this objective, we found that induction by p-coumaric acid enhances pesticide detoxification and tolerance and that consumption of honey enhances tolerance of aflatoxin B1 relative to consumption of high fructose corn syrup.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Schuler, M.A. and M.R. Berenbaum. Structure and function of cytochrome P450s in insect adaptation to natural and synthetic toxins: Insights gained from molecular modeling. Journal of Chemical Ecology 39: 1232-1245.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Berenbaum, M.R. Bees in crisis: Colony collapse, honey laundering, and other problems bee-setting American apiculture. Proc. Am. Phil. Soc.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Mao, W., M.A. Schuler and M.R. Berenbaum. Honey constituents upregulate detoxification and immunity genes in the western honey bee Apis mellifera. Proceedings of the National Academy of Sciences 110: 8842-8846.
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Progress 02/29/12 to 02/27/13
Outputs
Target Audience: Members of the target audience include insect toxicologists, physiologists, and sociobiologists (at meetings and professional societies), beekeepers and others in the apicultural community (through meetings of beekeeping societies), and the general public (through a variety of public meetings, presentations, and the like). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training activities include providing guidance and instruction to graduate student Catherine Dana on conducting bioassays of pesticide impacts on honey bees as well as guidance and instruction to graduate student Ling-Hsiu Liao on HPLC- and GC-based characterization of phytochemical metabolites. In addition, graduate student Mark Demkovich mastered methods for expressing honey bee P450s in a baculovirus-mediated expression system in order to quantify enzymatic activities of specific CYP9Q enzymes toward pesticides and phytochemicals and graduate studentKatherine Noble received training in annotating genomic and transcriptomic data. How have the results been disseminated to communities of interest? Results have been disseminated through scholarly publications and public presentations as discussed in the Other Products section of this report. What do you plan to do during the next reporting period to accomplish the goals? For Objective One, during the next reporting period we will continue to examine inducers and inhibitors and expand our focus to include propolis. For Objective Two,as we characterize phytochemicals we will continue to assay their impat on P450 detoxification; moreover, we will test for synergistic and antagonistic interactions with agricultural pesticides. For Objective Threewe will attempt to isolate and characterize CYP6BD1, which is upregulated by p-coumaric acid along with known xenobiotic detoxification enzymes CYP6AS1-4 and CYP9Q1-3. For Objective Fourif funding permits we will pursue RNASeq to characterize regulatory responses of different carbohydrate sources on detoxification and immunity genes and continue to conduct bioassays evaluating impacts of food sources on pesticide tolerance.
Impacts
What was accomplished under these goals?
With respect to Objective One -"To identify inducers and inhibitors of cytochrome P450 detoxification gene transcription in naturally occurring (nectar, pollen) and processed (honey, beebread) components of the honey bee diet" we successfully purified and identified four constituents of honey---p-coumaric acid, pinocembrin, pinobanksin and pinobanskin 5-methyl ether, as effective inducers of CYPQ detoxification enzymes. WIth respect to Objective Two -"To determine how in-hive and agricultural pesticides induce or inhibit P450 detoxification"these experiments are ongoing; our current focus is on the impacts of the newly approved acaricide amitraz on honey bee P450-mediated detoxification. With respect to Objective Three - "To identify specific P450 enzymes that metabolize pesticides and phytochemicals"this objective was accomplished in the previous reporting year; CYP9Q1-3 metabolize tau-fluvalinate, coumaphos and the agricultural pesticide bifenthrin, in addition to the honey flavonoid quercetin. RNASeq with p-coumaric acid also identified one additional candidate P450 gene (CYP6BD1) as a potential xenobiotic metabolizing P450 locus as well as an esterase gene. WIth respect to Objective Four - "To test whether diet (honey, sucrose, high fructose corn syrup) influences transcription of P450 genes and toxin tolerance" we confirmed that in vivo consumption of p-coumaric acid (in honey) enhances detoxification of the acaricide coumaphos. We also tested the efficacy of multiple nectar flavonoids as inducers of P450 activity.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Mao, W, MA Schuler and MR Berenbaum, Honey constituents upregulate detoxification and immunity genes in the western honey bee Apis mellifera. Proceedings of the National Academy of Sciences 110: 8842-8846. doi: 10.1073/pnas.1303884110.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Schuler, MA and MR Berenbaum, Structure and function of cytochrome P450s in insect adaptation to natural and synthetic toxins: Insights gained from molecular modeling. Journal of Chemical Ecology.
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: Objectives of this project (and experimental approaches) are: 1. Identify inducers and inhibitors of P450 gene transcription in natural substances (nectar, pollen) and hive products (honey, beebread, propolis) using quantitative real-time PCR followed by GC-MS/LC-MS to identify specific inducers/inhibitors. 2. Identify in-hive and agricultural pesticides that induce or inhibit P450 gene transcription (using qPCR). 3. Determine substrates for enzymes encoded by upregulated genes using baculovirus-mediated expression and molecular modeling. 4. Test whether inducers from hive products incorporated can hence enhances toxin tolerance using pesticide LD50 bioassays with and without inducers. Dissemination of these findings has taken place via radio interviews and personal presentations, including: "Honey and health," Illinois State Beekeepers Association, Champaign, IL June 25, 2011; WILL AM 580 Afternoon Magazine, Pollinatarium programs, August 5, 2011; UI Pollinatarium, Colony Collapse Disorder update, August 7, 2011; WILL AM 580 Focus 580, Bees in the news; ABC News bee stings August 26 2011; Chicago Tribune CCD September 1, 2011; Big Ten Housing Conference, "Honey and housing," Urbana, IL, October 25, 2011; Honey chemistry, Peggy Notebaert Museum, October 29, 2011; "History of honey," Garfield Park Conservatory Beekeeping Workshop, Garfield Park Conservatory, Chicago, IL, November 19, 2011; Fourth Dimension Fourth Presbyterian Church, Chicago, IL January 13, 2012; Southern Illinois Audubon Society, Carbondale, IL, January 27, 2012; interview for Naomi Klein January 31, 2012; "Beespotter," MSTE Lunch, Champaign, IL, February 17, 2012; "Bees and pesticides," Missouri State Beekeepers Association, St. Louis, MO, March 9, 2012; Bees and pesticides, UIUC Beekeeping Short Course, Urbana, IL, March 31, 2012; and "Honey chemistry," Backyard Beekeepers Association, Weston, CT, May 29, 2012. PARTICIPANTS: Wenfu Mao is a postdoctoral associate on this project learning and applying methods of molecular genetic analysis to bee toxicology. Catherine Dana is a graduate student learning how to conduct bioassays of behavioral responses of honey bees to pesticides. Katherine Noble is a graduate student learning how to conduct bioassays of behavioral responses of honey bees to p-coumaric acid and other constituents of honey, beebread and propolis. Ling Hsiu Liao is a graduate student studying how honey processing changes the phytochemistry of honey and how esterase activity contributes to xenobiotic detoxification in honey bees. Mark Demkovich is a graduate student learning how to express honey bee P450s in a heterologous system in order to assay possible synergistic interactions among pesticides. Brendan Colon is an undergraduate who is learning how to model molecular structure of xenobiotic-metabolizing cytochrome P450s in honey bees to determine mechanisms of synergistic interactions. TARGET AUDIENCES: Target audiences include insect toxicologists, physiologists, and sociobiologists (at meetings of professional societies), beekeepers and others in the apicultural community (through meetings of beekeeping societies), and the general public (through a variety of public meetings, presentations, and the like) PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the miticides tau-fluvalinate and coumaphos, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet in tau-fluvalinate bioassays no induction occurred in bees fed phenobarbital. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. Specialty microarrays containing all bee P450s revealed no change in detoxicative gene expression in guts of phenobarbital-treated bees. However, Northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450s. In bioassays, diet did not influence tau-fluvalinate or imidacloprid toxicity but aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey. We also used high pressure liquid chromatography, MS-MS, and bioassays to identify several honey constituents that act as inducers of detoxification gene transcription. These include pinobanksin, pinobanksin 5 methyl ether, pinocembrin, and p-coumaric acid; whereas the first three likely arise from propolis, p-coumaric acid is a universal constituent of pollen and is likely present in all honey and bee bread. Via RNAseq analysis, we demonstrated that p-coumaric acid specifically upregulates detoxification genes and select antimicrobial genes. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honeybees and may function as a nutraceutical regulating immune and detoxification processes. This finding confirms that the widespread apicultural use of honey substitutes, including high fructose corn syrup, may compromise the ability of honeybees to resist pesticides and pathogens and contribute to colony losses. As well, this finding raises uncertainties as to the applicability of previous studies of pesticide toxicity which were conducted with sucrose, rather than honey, as the delivery medium 3. Studies of behavioral responses to p-coumaric acid in addition to bioassays of p-coumaric acid impacts on pesticide toxicity 3. Additional bioassays of herbicides, insecticides and fungicides found in wax comb, bee bodies, or beebread are being conducted to determine whether these compounds interact synergistically. In addition to bioassays, molecular models are under construction to determine if these compounds interfere with each other in the catalytic site of the principal detoxificative enzymes (CYP9Q).
Publications
- Niu, G., Johnson, R.M. and Berenbaum, M.R. 2011. Toxicity of mycotoxins to honeybees and its amelioration by propolis. Apidologie: 10.1051/apido/2010039
- Johnson, R.M., Mao, W., Pollock, H.S., Niu, G., Schuler, M.A. and Berenbaum, M.R. 2012. Ecologically appropriate xenobiotics induce cytochrome P450s in Apis mellifera. PLoS ONE 7(2): e31051. doi:10.1371/journal.pone.0031051.
- Mao, W., Schuler, M.A. and Berenbaum, M.R. 2012. Honey bees rely on honey constituents to upregulate detoxification and immunity genes. For submission to Science.
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