Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema , and Crithidia Charles Runckel1,2., Michelle L. Flenniken3., Juan C. Engel4, J. Graham Ruby1,2, Donald Ganem1,2, Raul Andino3, Joseph L. DeRisi1,2* 1 Howard Hughes Medical Institute, Bethesda, Maryland, United State of America, 2 Departments of Medicine, Biochemistry and Biophysics, and Microbiology, University of California San Francisco, San Francisco, California, United States of America, 3 Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California, United States of America, 4 Sandler Center for Drug Discovery and Department of Pathology, University of California San Francisco, San Francisco, California, United States of America Abstract Honey bees (Apis mellifera) play a critical role in global food production as pollinators of numerous crops. Recently, honey bee populations in the United States, Canada, and Europe have suffered an unexplained increase in annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Epidemiological analysis of CCD is confounded by a relative dearth of bee pathogen field studies. To identify what constitutes an abnormal pathophysiological condition in a honey bee colony, it is critical to have characterized the spectrum of exogenous infectious agents in healthy hives over time. We conducted a prospective study of a large scale migratory bee keeping operation using high-frequency sampling paired with comprehensive molecular detection methods, including a custom microarray, qPCR, and ultra deep sequencing. We established seasonal incidence and abundance of known viruses, Nosema sp., Crithidia mellificae, and bacteria. Ultra deep sequence analysis further identified four novel RNA viruses, two of which were the most abundant observed components of the honey bee microbiome (,1011 viruses per honey bee). Our results demonstrate episodic viral incidence and distinct pathogen patterns between summer and winter time-points. Peak infection of common honey bee viruses and Nosema occurred in the summer, whereas levels of the trypanosomatid Crithidia mellificae and Lake Sinai virus 2, a novel virus, peaked in January. Citation: Runckel C, Flenniken ML, Engel JC, Ruby JG, Ganem D, et al. (2011) Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia. PLoS ONE 6(6): e20656. doi:10.1371/journal.pone.0020656 Editor: Robin F. A. Moritz, Martin-Luther-Universita¨t Halle, Germany Received March 30, 2011; Accepted May 6, 2011; Published June 7, 2011 Copyright: 2011 Runckel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a research grant from Project Apis m. (www.projectapism.org) awarded to J.D.R. J.D.R. is supported by the Howard Hughes Medical Institute. C.R. is supported by at the Genentech Graduate Student Fellowship. M.L.F. is supported by an A.P. Giannini Foundation Medical Research Fellowship and research funds from UC-Davis’s Ha¨agen Dazs Postdoctoral Fellowship in Honey Bee Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: joe@derisilab.ucsf.edu . These authors contributed equally to this work. Introduction Western honey bees (Apis mellifera) are highly social insects that live in colonies of ,30,000 individuals [1,2]. Honey bees are essential pollinators of agriculturally important crops including apples, almonds, alfalfa, and citrus. Current agricultural practices, such as large-scale monocultures, demand a seasonal abundance of honey bees in geographic locations incapable of maintaining sufficient pollinator populations year-round. Migratory beekeeping operations fulfill this need. For example, each February in the Central Valley of California 1.3 million honey bee colonies (,50% of the U.S. honey bee population) are required for almond pollination [3,4,5]. Pollination of this and other U.S. crops is valued at ,$15 billion annually [5]. There are numerous threats facing honey bee populations and the recent losses of honey bee colonies in the United States, Canada, and Europe is alarming. In the U.S., annual honey bee colony losses increased from 17–20% to 32% during the winter of 2006/07 with some operations losing 90% of their hives [6]. Average annual losses have remained high, averaging 32.6% from 2007–2010 [6,7,8]. One factor contributing to increased losses is Colony Collapse Disorder (CCD), an unexplained loss of honey bee colonies fitting a defined set of criteria [9,10]. While factors such as pesticide exposure, transportation stress, genetic diversity, and nutrition affect colony health, the most significant CCD- associated variable characterized to date is increased pathogen incidence [10]. Although greater pathogen incidence correlates with CCD, the cause is unknown in part due to insufficient knowledge of the pathogenic and commensal organisms associated with honey bees [10,11]. Parasitic threats to honey bee colonies include viruses, Nosema, bacteria, and Crithidia. The majority of honey bee infecting viruses are positive-sense single-stranded RNA viruses of the Picornavirales order. They include acute bee paralysis virus (ABPV) [12], black queen cell virus (BQCV) [13], Israeli acute bee paralysis virus (IAPV) [14], Kashmir bee virus (KBV) [15], deformed wing virus (DWV) [16], sacbrood virus (SBV) [17], and chronic bee paralysis virus (CBPV) [18] (reviewed in Chen and Siede, 2007 [19]). PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20656