Microbial  References – October 29, 2010

Laurie Ramona Herboldsheimer

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978 407 3934





“Microbes ‘R’ Us”, Judson O, July 22, 2009, The New York Times






“No Bee is an Island”, Stiglitz D, Herboldsheimer L, May 19, 2008



Gilliam, Martha (1997) Identification and roles of non-pathogenic microflora associated with honey bees



KaCaniova M, Chilebo R, Kopernicky M, Trakovicka A (2003) Microflora of the Honeybee Gastrointestinal Tract


(pay to download)


Cano RJ, Borucki MK, Higby-Schweitzer M, Pionar HN (1994) Bacillus DNA in Fossil Bees: an Ancient Symbiosis?



Microbiology of the insect gut: tales from mosquitoes and bees



Olofsson T, Vasquez A (2008) Detection and Identification of a Novel Lactic Acid Bacterial Flora Within the Honey Stomach of the Honeybee Apis Mellifera


(pay to download)


Forsgren E, Olofsson T, Vasquez A, Fries I (2009) Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae



Gilliam, Martha (1990) Chalkbrood Disease of Honey Bees, Apis Mellifera, Caused by the fungus, Ascosphaera apis: A Review of Past and Current Research

(available from USDA Carl Hayden Bee Lab, Tucson, Arizona)


Evans JD, Armstrong TN (2006) Antagonistic interactions between honey bee bacterial symbionts and implications for disease



Reynaldi FJ, Degusti MR, Alippi AM (1990) Inhibition of the Growth of Ascosphaera apis by Paenibacillus strains isolated from honey



Johnson RN, Zaman MT, Decelle MM, Siegel AJ, Tarpy DR, Siegel EC and Starks PT (2004) Multiple micro-organisms in chalkbrood mummies: evidence and implications



Starks PT, Blackie CA, Seeley TD (2000) Fever in honeybee colonies



Biodiversity and ecophysiology of yeasts / Carlos Rosa, Gabor Peter (eds.). Berlin; London: Springer 2006






Wickelgren I, “Scientist solves secret of bee bread”, Science News, 1988



Loper GM, Standifer LN, Thompson MJ and Gilliam M (1980) Biochemistry and Microbiology of Bee-Collected Almond (Prunus dulcis) Pollen and Bee Bread



Gilliam M, Prest DB, Lorenz BJ (1988) Microbiology of pollen and bee bread: taxonomy and enzymology of molds



Gilliam, Martha (1979) Microbiology of pollen and bee bread: the yeasts



Gilliam, Martha (1979) Microbiology of pollen and bee bread: the genus Bacillus



Reynaldi FJ, Degusti MR, Alippi AM (1990) Inhibition of the Growth of Ascosphaera apis by Paenibacillus strains isolated from honey






Wheeler, William Morton (1907) The Fungus-Growing Ants of North America



Live-in Domestics: Mites as Maids in Tropical Rainforest Sweat Bee Nests (April 2009)



“Gut Reactions”, Margonelli  L, The Atlantic Magazine, September 2008







Gilliam M, Prest D, Morton H (1974) Fungi isolated from honey bees, Apis mellifera, fed 2, 4-D and antibiotics



Gilliam M, Morton H (1973) Enterobacteriaceae isolated from honey bees, Apis mellifera, treated with 2,4-D and antibiotics



Gilliam M, Wickerham J, Morton H, Martin R (1974) Yeasts Isolated from Honey Bees, Apis Mellifera, Fed 2, 4-D and Antibiotics



Gilliam M, Morton H (1977) The Mycroflora of Adult Worker Honeybees, Apis Mellifera: Effects of 2,4,5-T and Caging of Bee Colonies



Yoder J, Christensen B, Croxall T, Tank J (2008) Suppression of Growth Rate of Colony-Associated Fungi by High Fructose Corn syrup Feeding Supplement, Formic Acid, and Oxalic Acid



Jakobsons Boriss (2005) Biological treatment of chalkbrood in honey bees



Honey Bee Nutrition & Medication.  The Consequences of Decades of Quick Fixes.  Guest Editorial by Bruce Brown, Bee Culture magazine, April 2008


Kubik M, Nowacki J, Pidek A, Warakomska Z, Michalczuk L, Goszczynski W (1999) Pesticide residues in bee products collected from cherry trees protected during blooming period with contact and systemic fungicides



Kubik M, Nowacki J, Pidek A, Warakomska Z, Michalczuk L, Goszczynski W, Dwuznik B (2000) Residues of captan (contact) and difenoconazole (systemic) fungicides in bee products from an apple orchard



vanEngelsdorp D, Evans J, Donovall L, Mullin C, Frazier M, Frazier J, Tarpy D, Hayes J, Pettis J  (2009) “Entombed Pollen”: A new condition in honey bee colonies associated with increased risk of colony mortality



Mullin C, Frazier M, Frazier J, Ashcroft S, Simonds R, vanEnglesdorp D, Pettis J (2010) High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health



“Fungicides can reduce, hinder pollination potential of honey bees”, Western Farm Press, March 7, 2009



The Treevine, Gary Gliddon, February 2010, Volume 18, issue 2



PAN Pesticides Database – California Pesticide Use

Pesticide Use on Almonds in 2008




“Beekeepers and Biochemistry” from Eric Mussen’s newsletter



USDA/ARS Research Project: Determining the Impact of Pristine on Bee Fungus



USDA/ARS Research Project: Accessing Microbial Activity in Honey Bees




Abstracts (from yet to be published papers) from the Pesticide-Pollinator Workshop, July 22 2010, Alfred Sate College, Alfred, NY:


The effects of fungicides on the diversity of microbes in stored pollen and the physiological

repercussions on worker and queen honey bees

Gloria DeGrandi-Hoffman, Kirk Anderson, Mark Carroll, Bruce Eckholm, and Diana

Sammataro, Carl Hayden Bee Research Center, USDA-ARS, 2000 East Allen Road, Tucson, AZ


“Honey bee colonies harbor a wide range of microbes, many of which play vital roles in the

preservation and digestion of pollen. Bees store pollen in comb cells and it is there that the pollen is fermented through the action of microbes and converted into bee bread. There are numerous bacteria and fungi present in bee bread that pre-digest the pollen grains and make the nutrients inside more accessible to the bees. The microbes also supply essential nutrients through their metabolic processes. The action of symbiotic microbes might be compromised if they are exposed to pollen contaminated with fungicides. To test this, we collected pollen from colonies in almond orchards during pollination. In this pollen we detected >6000 ppb of Boscalid, 1700 ppb of Pyraclostrobin, >2800 ppb of Propiconazole, and >12500ppb of Iprodione. Bee bread sampled from colonies in the same orchard had >9000 ppb of Boscalid, >2000ppb of

Pyraclostrobin, and 7700 ppb of Iprodione. Propiconazole was not detected in the bee bread

samples. From our pilot studies, we found that bee bread made from pollen contaminated with

fungicides has a lower diversity of microbes compared with bee bread made from  uncontaminated pollen. In our current work, we are investigating the effects of the reduction in

microbial diversity on the ability of bees to process pollen into worker jelly. Whether there are

effects on the ability of the queen to lay eggs and generate volatile signals communicating her

egg laying activity also is being determined.”


Drug interactions between miticides and fungicides in honey bees (Apis mellifera)

Reed M. Johnson, Department of Entomology, University of Nebraska-Lincoln, Lincoln, NE


The ectoparasitic Varroa mite (Varroa destructor) is one of the most serious pests of honey bees today. Beekeepers often suppress Varroa infestations using pesticides applied at therapeutic doses as anti-parasitic drugs. Three commonly used synthetic miticidal drugs – coumaphos (Checkmite+TM), fenpyroximate (HivastanTM) and tau-fluvalinate (ApistanTM) – appear to be tolerated through cytochrome P450 (P450) mediated detoxification in bees. Just as metabolic interactions can occur between drugs in humans, drug interactions can also occur between miticides detoxified by P450s in bees. Simultaneous exposure to multiple miticides is likely to occur given the high levels of miticide contamination reported in beeswax. Bees are also likely to be be exposed to high doses of fungicides applied to bee-pollinated crops. Fungicides are generally considered safe for bees and there are few restrictions on their application during bloom. However, some fungicides may affect bees' ability to tolerate miticides. Chlorothalonil (BravoTM), a common fungicide found in pollen stores and wax, decreases bees' tolerance of mitides. Prochloraz, an inhibitor of cytochrome P450 activity in fungi, increases the toxicity of coumaphos, fenpyroximate, and greatly increased the toxicity of tau-fluvalinate. Based on these findings it would be prudent for beekeepers to avoid repeated use of P450-interacting miticides and to avoid using these miticides when bees are likely to come into contact with these or other potentially interacting fungicides.


Sub-lethal pesticide exposure in honey bees: Chronic pesticide exposure at the colony level

results in increased susceptibility of workers to pathogen infection

Jeffery S. Pettis, USDA-ARS Bee Research Laboratory, Beltsville, MD 20705 USA

Dennis vanEngelsdorp, Penn State University, Department of Entomology, University Park, PA

Josephine Johnson, University of Maryland Baltimore, Department of Toxicology, Baltimore,


Galen Dively, University of Maryland, Department of Entomology, College Park, MD

Global pollinator declines have been attributed to habitat destruction, pesticide use and climate change or some combination of these factors and managed honey bees, Apis mellifera, are part of worldwide pollinator declines. We exposed honey bee colonies during three brood generations to sub-lethal doses of a widely used pesticide, imidacloprid, and then subsequently challenged newly-emerged bees with the gut parasite, Nosema ceranae. The pesticide dosages used were below levels that are thought to cause harm to honey bees. We demonstrated an increase in pathogen growth within individual bees reared in colonies exposed to the pesticide. Interactions between pesticides and pathogens could be a major contributor to increased mortality of honey bee colonies and other pollinators worldwide. Other sub-lethal pesticides studies will also be discussed in trying to understand the role of pesticides in declining bee health.


Pesticides and Pollinators: Assessing Residues and Multiple Interactions in Honey Bees

Chris Mullin, James L. Frazier and Maryann Frazier, Department of Entomology, The

Pennsylvania State University, University Park, PA 16802

Honey bees encounter numerous toxic chemicals through hive treatments and on foraging trips to acquire essential pollen and nectar foods. Recently, a broad survey for pesticide residues was

conducted in pollen, wax, bee and associated hive samples in proximity to diverse fruit, nut,

vegetable and seed cropping sites in US and Canada. We found 121 different pesticides and

metabolites up to 214 ppm using a modified QuEChERS method and LC/MS-MS and GC/MS.

The fungicide chlorothalonil and both in-hive miticides fluvalinate and coumaphos co-occurred

in 47% of wax and pollen samples, while the 3 were combined with a systemic pesticide (e.g.

aldicarb, boscalid, imidacloprid, and myclobutanil) in 32% of samples. There were fewer

pesticides, particularly systemics, found in bees except for those linked to bee kills. Almost all

comb and foundation wax was contaminated with miticides and other pesticides, averaging 8

detections with a high of 39 per sample. Synergistic effects on bees were established for the

contact fungicide chlorothalonil in combination with miticides and systemic insecticides and

fungicides frequently coincident in pollen. Potential consequences to bee health of pesticide

combinations in their food and comb, including pro-systemicides and their toxic degradates, will be discussed.



Abstracts (from yet to be published papers) from the Association of Southeastern Biologists annual meeting April 7-10, 2010, Asheville, NC




P2.59 BRADY S. CHRISTENSEN1, TRAVIS J. CROXALL1, JAY A. YODER1, DIANA SAMMATARO2 AND GLORIA DeGRANDI-HOFFMAN2. Wittenberg University1, USDA-ARS, Carl Hayden Honey Bee Research Center2.    Spraying fungicides reduces symbiotic microbes necessary for bee bread production.

Honey bee (Apis mellifera) development depends on fungal conversion of stored pollen into bee bread that is fed to larval bees. A combination field-mycological study was done surveying 21 hives in orchards representing various levels of fungicide treatment to determine the amount of fungi present and affected in bee bread. All bee bread samples are characterized by a regular mycoflora profile dominated by Aspergillus spp. and Penicillium spp. and to a lesser extent Cladosporium spp. and Rhizopus spp. Minor components were Alternaria spp., Aureobasidium spp., Bipolaris spp., Colletotrichum spp., Fusarium spp., Mucor spp., Paecilomyces spp., Scopulariopsis spp., Stigmella spp. and Trichoderma spp. (mixed composition), presumably reflective of habitat differences. Bee colonies in direct fungicide spraying resulted in an overall decrease of all fungal components, not a select group or single kind of fungus. This decline correlated with a 3-4 fold suppression in conidia production, 16 hours or 68 hours after spraying. Even if not sprayed with fungicide directly, colonies within 3.2km bee flight range of sprayed areas showed similar reductions in fungal loads as observed in bee bread from directly sprayed areas. Surprisingly, this included colonies from an organic orchard. We conclude that direct and indirect fungicide exposure is disrupting the bee colony fungal community, with implications for death by production of nutritionally-poor food. Beekeepers report increased incidence of chalkbrood disease after fungicide spraying that we now attribute to the pronounced reduction of Aspergillus spp. and Penicillium spp. that are inhibitory toward bee pathogens.


P2.60 BRIAN Z. HEDGES1, DAVID M. KOLAKOWSKI1, JAY A. YODER1, DIANA SAMMATARO2 AND GLORIA DeGRANDI-HOFFMAN2. Wittenberg University1, USDA-ARS, Carl Hayden Honey Bee Research Center2. Alteration of honey bee (Apis mellifera) colony nutritional source, "bee bread", in response to fungicide exposure.

Symbiotic bee colony fungi convert stored pollen into bee bread, satisfying an absolute dietary requirement for developing bee larvae. When sprayed, fungicides are brought into the colony by bees via contaminated pollen. This study explores effects of fungicide on bee bread fungi in vitro by radial growth rate determination of 12 bee bread fungal isolates with Pristine® (BASF), a broad spectrum fungicide frequently applied to various commercial crops. Natural comb cell conditions were simulated by conducting the experiment on bee-bread supplemented non-nutritive agar, 30oC, darkness, and 5% CO2. Radial growth rates for each fungus were characteristic and were reduced 12% - 80% by fungicide, depending on species and concentration, in a dose-response. Percentage reduction in growth rates, mortality, and least effective concentration differed among the 12 fungi and did not correlate with whether the fungus was a slow/moderate- or fast-grower; i.e., no two fungi responded the same. Effectiveness of Pristine is species (likely strain)-specific and is not a function of slow growth retaining fungi on treated surfaces longer or decreased exposure times by faster growers that spread rapidly. Most tolerant fungi to Pristine were Rhizopus sp., Mucor sp., and Absidia sp., and Penicillium sp. and Aspergillus niger were the most sensitive. Pristine had a controlling effect on bee fungal pathogens, Ascosphaera apis (chalkbrood) and Aspergillus flavus (stonebrood). Thus, bee bread fungi respond to fungicide differently and could have a negative effect on colony health by altering the composition of mycoflora that bees use to process and store their food.



P2.61 DERRICK J. HEYDINGER1, MICHAEL R. CONDON1, JAY A. YODER1, DIANA SAMMATARO2 AND GLORIA DeGRANDI-HOFFMAN2. Wittenberg University1, USDA-ARS, Carl Hayden Honey Bee Research Center2. Commercially applied antibiotics are ineffective against honey bee diseases chalkbrood and stonebrood.

Fumagillin (Fumagilin-B®, Medivet), tylosin (Tylan®, Eli Lilly) and oxytetracycline (Terramycin®, Pfizer) are applied to control nosemosis (Nosema apis) and foulbrood (Paenibacillus larvae) in honey bee, Apis mellifera, colonies. The purpose of this study was to explore whether these antibiotics alter the growth of bee breed fungi that convey colony defense (antibiotic producers), provide a source of digestive enzymes in adult bees, and make food for bee larvae from stored pollen as a developmental requirement. Based on fungus culturing, the trisecting line method was used to determine radial growth rates of the 13 most frequently recovered bee fungal isolates, including Ascosphaera apis (agent of chalkbrood disease) and Aspergillus flavus (agent of stonebrood disease), on media treated with antibiotics (1%, 0.1%, 0.01%), alone and in combination. To mimic conditions for making bee bread in a capped wax

cell, we measured fungal growth on agar supplemented only with bee bread nutrients at 30oC in darkness and transferred from 5% CO2 to aerobic conditions. Under these conditions, antibiotic exposure produced no changes in obverse/reverse pigmentation, colony, conidia, philiade characteristics, or initiated production of teleomorphs in any of the 13 fungi. No fungi displayed antibiotic sensitivity, evidenced by lack of dose-response, mortality, antibiotic synergistic effects, and difference from control growth rates. These results suggest that shifts in composition of the bee colony mycoflora are unlikely to occur by use of these antibiotics. Important information for commercial beekeepers is that these tested antibiotics are not effective treatment against stonebrood and chalkbrood.