Several DNA viruses that infect honey bees have also been described [19]. Viral infections in bees can remain asymptomatic, or cause deformities, paralysis and/or death [19,20]. Symptoms associated with specific viruses include wing deformities (DWV), hairless, dark, shiny bees (CBPV), swollen yellow larva and/or dark-brown larva carcasses in the cells of worker-bees (SBV) or queen-bees (BQCV), however accurate diagnosis requires molec- ular biology techniques as asymptomatic bees frequently test positive for one or more viruses [19,21,22,23]. In addition to viral infections, honey bees are also readily parasitized by the microsporidia Nosema [19,24]. Historically U.S. honey bees were predominantly infected by Nosema apis, but recently Nosema ceranae infections dominate [24,25]. The effects of Nosema infection on individual bee and colony health are unclear [24,26]. Some reports suggest infections decrease longevity and may lead to collapse [27,28,29], but since Nosema is widespread and often detected in healthy colonies its role in colony health requires further investigation [10,26,30]. Another fungal pathogen Asco- phaera apis, the causative agent of Chalkbrood disease, kills infected larvae, but does not typically cause colony loss [31,32]. Bacterial pathogens of honey bees include Paenibacillus larvae and Melissococcus plutonius, the causative agents of American and European Foulbrood disease [33,34,35,36]. In addition to microbial infections, mite infestation (Ararapis woodi, Tropilaelaps sp., and Varroa destructor) also weakens and kills honey bee colonies [37,38]. Introduction of V. destructor mites, which feed on the hemolymph of developing honey bees and transmit viruses (DWV, KBV, IAPV), in the late 1980s was devastating to the U.S. honey bee population [39,40,41]. Notably, the restricted genetic diversity of the U.S. honey bee population may make it particularly susceptible to catastrophic and episodic losses [42,43]. To gain a more complete understanding of the spectrum of infectious agents and potential threats found in commercially managed migratory honey bee colonies, we conducted a 10-month prospective investigation. Our broad-scale analysis incorporated a Temporal Analysis of the Honey Bee Microbiome suite of molecular tools (custom microarray, polymerase chain reaction (PCR), quantitative PCR (qPCR) and deep sequencing) enabling rapid detection of the presence (or absence) of all previously identified honey bee pathogens as well as facilitating the detection of novel pathogens. This study provides a comprehensive temporal characterization of honey bee pathogens and offers a baseline for understanding current and emerging threats to this critical component of U.S. agriculture. Results Following devastating losses suffered by U.S. commercial beekeeping operations in 2006–2007, we initiated a prospective study monitoring a typically managed, large-scale (.70,000 hives), migratory commercial beekeeping operation over 10-months. Honey bees from 20 colonies were consistently sampled beginning with the introduction of a new queen in April 2009 (Mississippi (MS), through transport to summer foraging grounds in South Dakota (SD), and transfer to California (CA) for almond pollination (Figure 1). During our study, these colonies were exposed to antimicrobial treatments, transportation stress, differ- ent pollen and nectar sources, and three distinct geographic locations: MS, SD, and CA, (U.S.A.). A molecular analysis pipeline consisting of custom microarray, polymerase chain reaction (PCR), quantitative PCR (qPCR) and ultra deep sequencing was employed to characterize the honey bee microbial flora. Pathogen screening was performed using the ‘‘Arthropod Pathogen Microarray’’ (APM) built on the same design principles used for human pathogen microarray screening [44,45]. The array’s design couples highly-conserved nucleic acid targets with hybridization-based detection to identify previously uncharacterized organisms [46,47,48,49,50,51]. Specifically, the APM was designed to detect virtually all known microbial parasites of insects. Endpoint PCR provided sensitive detection while qPCR documented abundance of select pathogens. Ultra Figure 1. Temporal monitoring of the honey bee microbiome from 20 monitor colonies within a large-scale migratory U.S. beekeeping operation using a custom arthropod pathogen microarray, PCR, quantitative PCR, and ultra deep sequencing. The colonies were established with new queens in Mississippi (MS) in April 2009, moved to South Dakota (SD) in May 2009, and finally to California (CA) in November 2009; monitoring concluded in January 2010. doi:10.1371/journal.pone.0020656.g001 PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20656