deep sequencing facilitated the discovery of novel and highly divergent microbes. Together the results from our monitoring study provide insight regarding the incidence of virus and microbe infections in honey bee colonies. Arthropod Pathogen Microarray design and validation The Arthropod Pathogen Microarray (APM) is a custom DNA microarray capable of detecting over 200 arthropod associated viruses, microbes, and metazoans. This DNA microarray includes oligonucleotides representing every arthropod-infecting virus with published nucleic acid sequence in the International Committee on Taxonomy of Viruses database as of November 2008 [52,53]. Design principles used for APM oligonucleotides (70-mers) were based on previous pan-viral microarrays using ArrayOligoSelector (AOS) [54]. In addition, non-viral pathogens including, Nosema (microsporidia), Crithidia (trypanosomatid), Varroa (mite), Tropilae- laps (mite) and Acarapis (tracheal mite) as well as Paenibacillus larvae and Melissococcus plutonius bacterial species [53] were represented on the microarray (Table 1). This new diagnostic tool is composed of 1536 oligonucleotides, including viral, non-viral and positive control targets (Table 1). Array analysis is performed computa- tionally using e-predict [54,55]. The sensitivity of the APM was estimated to be 1.96105 viral genome copies (1 pg Drosophila C virus in vitro transcribed genomic RNA) in an A. mellifera RNA (1 mg) background (see Materials and Methods). Array specificity was confirmed by performing pathogen-specific PCRs in conjunc- tion with nucleic acid sequencing. Test samples included honey bees from managed and feral colonies, Vespula sp. (yellow jackets), and Bombus sp. (bumble bees) (Table S1). A sample from a collapsed colony in Montana tested positive for the highest number of viruses (BQCV, DWV, KBV, IAPV) and documented the array’s ability to simultaneously detect multiple pathogens. Analysis of symptomatic honey bees, such as hairless, shiny bees and bees with deformed wings, confirmed the presence CBPV and DWV, respectively [56,57]. Likewise, analysis of Varroa destructor RNA validated the array’s ability to detect mites and their associated viruses (DWV). Interestingly, pathogens normally associated with honey bees, DWV and ABPV, were also detected in a yellow jacket sample (Vespula sp.) obtained near a hive entrance from which the honey bees also tested positive for ABPV and DWV. We used the APM to detect several pathogens (BQCV, DWV, SBV and Nosema) in CCD-affected colony samples from an Oklahoma based migratory beekeeping operation (Feb. 2009). In total we detected and sequence confirmed ten previously characterized honey bee pathogens using the array including: CBPV, IAPV, DWV, ABPV, BQCV, SBV, KBV, Nosema apis, N. ceranae and Varroa destructor. Temporal monitoring of 20 migratory honey bee colonies Honey bee samples were collected during their travels from Mississippi through South Dakota to California resulting in a prospectively collected 10-month time-course of 431 data points, each consisting of 50–100 bees isolated separately from both the entrance (older foragers) and brood comb (younger house bees). Hives #10, #14 and #19 were lost in December due to queen death or infertility. We analyzed all the entrance samples (5 bees per colony each time-point) using the APM. Nosema. There was an abundance of Nosema infections in our monitor colonies throughout the entire time-course. APM monitoring revealed that approximately half of the colonies in April and May were Nosema positive (Figure 2A). Notably, nearly every colony was infected during a surge in August and September. In order to determine which Nosema species was Temporal Analysis of the Honey Bee Microbiome Table 1. Oligonucleotide targets for the Arthropod Pathogen Microarray. Dicistrovirus acute bee paralysis virus black queen cell virus Israeli acute paralysis virus Kashmir bee virus other Dicistroviruses Iflavirus deformed wing virus honey bee slow paralysis virus sacbrood virus other Iflaviruses Other Virus Families Ascovirus Baculovirus Birnavirus Cypovirus Densovirus Idnoreovirus Iridovirus Luteovirus Nimavirus Nodavirus Okavirus Poxvirus Rhabdovirus Tetravirus Totivirus Unassigned Virus Families chronic bee paralysis virus Solenopsis Invicta virus II Acyrthospihon Pisum virus Nora virus kelp fly virus Bacteria Achromobacter Paenibacillus Melissococcus Enterococcus Wolbachia Brevibaccilus Fungi/Protists Crithidia Nosema Ascophaera Aspergillus Metarhizium Hirsutella Varroa Tropilaelaps Mites Total: 264 Total: 128 Total: 794 38 42 26 42 116 22 24 22 60 80 138 12 98 110 10 46 10 20 68 10 74 10 30 10 26 26 12 12 12 14 22 10 12 6 6 20 20 10 20 20 12 32 16 Total: 88 Total: 70 Total: 102 Total: 80 PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20656