for overnight shipment to our laboratory. Samples were stored at 280uC until RNA extraction; excess bees were archived for long- term 280uC storage. Time-course samples were collected monthly from April 15 (week 1) through July 14 (week 14), 2009 and weekly samples were attempted thereafter, however due to inclement weather or shipping logistics the samples for weeks 15, 28–30, 32, and 39–41 were not collected. A total of 864 samples were obtained and 431 exterior samples were analyzed. Honey bee sample preparation We determined that analysis of five honey bees per sample was sufficient for our colony monitoring project. Arthropod pathogen microarray (APM) analysis of test samples revealed that combined analysis of 5 bees reproducibly detected most, if not all, of the pathogens detected from 10 or 15 independently analyzed bees from the same sample. In addition, we confirmed the consistency of APM results by performing multiple analyses of a single RNA sample. Based on our test results and practical sample handling considerations, we reasoned that repeated analysis of 5 bees from each colony over-time (115 bees per colony) was sufficient for this study. Honey bee samples, 5 bees per colony each time-point, were homogenized in 1 mL 50% TRIzol Reagent (Sigma) and 50% phosphate buffered saline (PBS, UCSF Cell Culture) solution in a 2 mL micro-centrifuge tube containing one sterile zinc-coated steel ball bearing (5 mm) using a TissueLyzer II (Retsch), for 4 minutes at 30 Hz. RNA was isolated according to TRIzol Reagent (Invitrogen) manufacturer’s instructions. In brief, TRIzol reagent honey bee homogenate was combined with 0.1 ml chloroform and mixed by vortexing for 5 seconds, samples were incubated at room temperature for 5 minutes, prior to centrifu- gation for 10 minutes at 13,2006 g in a table top centrifuge. Next, 700 mL of the aqueous phase was transferred to a new microfuge tube containing 490 mL isopropanol. Following mixing, the samples were incubated at 220uC for 20 minutes and then either centrifuged (13,2006 g for 15 min) or further purified utilizing Zymo-III RNA columns according to manufacture’s instructions (Zymo). RNA was extracted from five bees collected from the colony entrance for each of the time-course samples. Arthropod Pathogen Microarray design and synthesis Design principles used for APM oligonucleotides (70 nt) were based on previous pan-viral microarrays using ArrayOligoSelector (AOS) [54]. Briefly, array oligonucleotides were selected for uniqueness against an insect nucleic acid background, for ,50% GC content to maintain high complexity, and for cross-reactivity of highly-conserved nucleic acid features with evolutionarily related targets (,250 kcal/mol predicted binding energy). Arthropod pathogen oligonucleotides (GEO GPL11490) were synthesized by Invitrogen, suspended at 40 pmol/mL in 36 SSC and 0.4 pmol/mL control oligo and printed on poly-L-lysine slides (Thermo) with silicon pins as previously described [100]. Each oligonucleotide and its reverse complement were printed twice for redundancy. Arrays were allowed to air-dry and stored and room temperature. Prior to use, oligonucelotides were cross-linked to slides via UV exposure (600 mJ), washed with 36 SSC/0.2% SDS and blocked using a methylpyrrolidone solution (335 mL 1- methyl-2-pyrrolidinone, 5.5 grams succinic anhydride, 15 mL 1 M sodium borate). Sample Preparation for Arthropod Pathogen Microarray (Reverse Transcription, CyDye Labeling, Hybridization, Scanning) For each sample, 5 mL (,15 mg nucleic acid) of extracted material was randomly primed and amplified as previously Temporal Analysis of the Honey Bee Microbiome described [44,45]. Briefly, an adapter-linked random nonamer (59GTTTCCCACTGGAGGATANNNNNNNNN) was used to prime the reverse transcription reaction using SuperScript II (Invitrogen). The same oligo is used for two rounds of second- strand synthesis with Sequenase (USB) in order to produce adapter-flanked sequences from both RNA and DNA starting material. One-quarter of the random priming reaction is used in a 50 mLTaq PCR reaction for 25 cycles with a single primer (59GTTTCCCACTGGAGGATA). One-tenth of the amplified material was further amplified for 10–20 cycles with a Cy3-linked primer (59Cy3 -GTTTCCCACTGGAGGATA). Samples were purified with the Zymo DNA Clean and Concentrator (Zymo) and resuspended in a buffer of 36 SSC, 50 mM HEPES and 0.5% SDS, and hybridized on the APM overnight at 65uC. Arrays were washed and scanned with an Axon 4000A scanner. Samples were analyzed manually and scored as positive for a pathogen if at least three unique oligonucleotides hybridized with at least five times background intensity. Arrays were further analyzed by a second unbiased method using the E-Predict algorithm [54,55], wherein all virus genomes were computationally hybridized to the array oligos and array results are compared to expected binding profiles. The top 5 unique oligos were removed and the algorithm reiterated twice in order to improve detection of low titer target(s) during a co-infection. Known honey bee pathogens were called positive if they exceeded a similarity score of 0.001 and were the highest ranked call in any iteration. In the event of a disagreement between the two analysis methods, a specific PCR reaction was performed, using material from the first PCR step, to resolve the call. Assessment of Arthropod Pathogen Microarray sensitivity In order to estimate the sensitivity of the arthropod pathogen microarray (APM) two positive control samples were prepared in the presence and absence of pathogen-free honey bee RNA. A full- length (9,264 nucleotide) Drosophila C virus (DCV) clone was in vitro transcribed, serially diluted into honey bee RNA, reverse- transcribed, amplified, dye-labeled and hybridized to the APM as described above. Detection of at least 3 of the 8 unique DCV oligonucleotides and their reverse complements resulted in an estimated DCV detection level of 1.96105 genome copies (1 pg DCV genomic RNA) in an A. mellifera RNA (1 mg) background. Similarly, detection of a BQCV genome segment (452 nt), corresponding to one array oligo and its reverse complement, diluted into either pathogen-free honey bee RNA (0.5 mg) or water indicated detection limits of 1.26105 genome segment copies (30 fg BQCV RNA segment) and 1.26104 genome segment copies (3 fg BQCV RNA segment) respectively. PCR Reaction conditions for polymerase chain reaction (PCR) amplifications of select samples were performed under the following conditions: 5 mL of 1:10 dilution of PCR-amplified DNA and 10 pmol of each forward and reverse primers were amplified with Taq polymerase with the following cycling conditions: 95uC for 5 min; 95uC for 30 s, 50–60uC for 30 s, 72uC for 1 min, 35 cycles; final elongation 72uC for 7 min, hold at 4uC. Select samples were Sanger sequenced directly from ExoI and SAP treated PCR product or from colony PCR of TOPO cloned (Invitrogen) gel-extracted bands. Bands produced by PCR assays for known honey bee pathogens were sequenced until each molecular weight product was unambiguously associated with either a true positive or non-target amplification of the honey bee genome or microbiome. All PCR results for the four novel viruses were confirmed by Sanger sequencing. PLoS ONE | www.plosone.org 13 June 2011 | Volume 6 | Issue 6 | e20656