30 s, 35 cycles; final elongation 72uC for 4 min, hold at 4uC. In addition to amplification and detection of the LSV replicative form using tagged-negative strand primed cDNA template and TAGS forward and LSVU-R1717 PCR primers, negative and positive controls were performed (Figure S5 – labeled (1)). Negative controls included utilizing unprimed RT reaction as a template for PCR amplification using TAGS forward and LSVU- R1717 primers (labeled (2)), LSV tagged negative-strand primed cDNA template in PCR reaction in which only the LSVU-R1717 primer was added in order to ensure that all of the unincorporated RT primer was digested with exonuclease I and thus not involved in priming the PCR reaction (labeled (5)), and no template PCR using LSV qPCR primer sets (labeled (6)). Positive controls included using random hexamer primed cDNA as template for PCR amplification using LSV1 or LSV2 -specific forward primer and LSVU-R-1717 (labeled (3)) and random hexamer primed cDNA amplified using LSV-specific qPCR primer sets (labeled (6)). PCR products were analyzed using agarose (2%) gel electrophoresis (Figure S5). Crithidia mellificae strain SF - Microscopy, Culturing and DNA Purification Honey bees were collected from a San Francisco, CA (U.S.A.) colony previously identified to be Crithidia positive by microarray and PCR testing. Honey bees were immobilized by chilling at 4uC for 20 minutes, briefly washed in 70% ethanol, and decapitated prior to dissection. The SF strain was isolated from honey bee intestines dissected in a sterile environment, minced and placed in a T25 flask and cultured in BHT medium composed of Brain Heart Infusion (BHI) 28.8 g/L (DIFCO), tryptose 4.5 g/L (DIFCO), glucose 5.0 g/L, Na2HPO4 0.5 g/L, KCl 0.3 g/L, hemin 1.0 mg/L, fetal bovine serum (heat inactivated) 2% v/v, pH 6.5, and containing penicillin G sodium (106 units/L) and streptomycin sulfate (292 mg/L) at 27uC [101]. Free active Crithidias were observed 24 hours post inoculation. Parasites were maintained by subculture passage every 4 days; stable liquid nitrogen stocks were archived. Light microscopy of live parasites was performed using a Leica DM6000 microscope equipped with Hamamatsu C4742-95 camera and Volocity Software (PerkinEl- mer). Imaging fixed parasites (4% paraformaldehyde, 20 min) facilitated visualization of DAPI (49,6-diamidino-2-phenylindole) stained nuclear and kinetoplast DNA. Images of fixed Crithidia mellificae were obtained using both the Leica DM6000 microscope and a Zeiss LSM 510-M microscope equipped with both a 636 objective numerical aperture 1.4, and a 1006 objective numerical aperture 1.4. For DNA purification, Crithidia mellificae (,106 trypanosomes/ mL culture medium) were pelleted by centrifugation (8006g for 6 min) and washed with PBS prior to DNA extraction. DNA was extracted using the DNeasy Genomic DNA Extraction Kit (Qiagen) as per the manufacturer’s instructions. Bees from Crithidia positive hives were homogenized by TissueLyser as above and DNA extracted using the DNeasy kit for the initial PCR screens, after suspension in either PBS or 16 Micrococcal Nuclease Buffer (NEB). Ultra Deep Sequencing Library Preparation Total nucleic acid from all twenty monitor hives at time-point 17 (August 5, 2009) was pooled (approximately 3 mg per hive). One quarter was treated with RNase A/T1 (Fermentas) and genomic DNA was isolated using a DNeasy column (Qiagen). 50 ng of genomic DNA was prepared for deep sequencing by Nextera recombinase (Epicentre) per the manufacturer’s instruc- tions. The remaining nucleic acid was treated with Turbo DNase Temporal Analysis of the Honey Bee Microbiome (Ambion) and column purified (Zymo) before being split into thirds. One third was enriched for mRNAs with dT-linked Dynabeads (Invitrogen). RNA from this fraction and from a second unenriched fraction were primed for RT and second- strand synthesis with an adapter linked oligo as above using oligo SolCommonN (59CGCTCTTCCGATCTNNNNNN). The third fraction of RNA was primed with an anchored oligo dT and subjected to two rounds of second strand synthesis with SolCommonN. Half of the initial material was amplified with primer SolCommon (59CGCTCTTCCGATCT) with KlenTaq (Sigma) at an annealing temperature of 37uC for 20 cycles. Reactions were cleaned by Zymo column, analyzed by NanoDrop spectrophotometer and 50 ng was used in a four-primer PCR reaction. In a 50 mL KlenTaq reaction, 10 pmol each of pri- mers 5Sol1 (59AATGATACGGCGACCACCGA) and 5Sol1 (59CAAGCAGAAGACGGCATACG) and 0.5 pmol of Sol1 (59 AATGATACGGCGACCACCGAGATCTACACTCTTTC- CCTACACGACGCTCTTCCGATCT) and Sol2 (59CAAGCA- GAAGACGGCATACGAGATCGGTCTCGGCATTCCTGC- TGAACCGCTCTTCCGATCT) were incubated for 2 cycles annealing at 37uC and 10 cycles at 55uC. Products were run on an 8% native acrylamide TBE gel (Invitrogen) and a 300–350 nt smear was cut out and electro-eluted. The product was further amplified at an annealing temperature of 55uC with primers 5Sol1 and 5Sol2 for 5–10 cycles until at least 30 ng of material was produced, as determined by NanoDrop. Libraries were sequenced on an Illumina Genome Analyzer II with a V3 cluster generation kit and V5 sequencing reagent as per the manufacturer’s instructions, producing paired-end 65 nt reads. Solexa Data Analysis and Virus Genome Recovery Six pools of sequence data were downloaded from GenBank: Nosema ceranae (draft genome), Spiroplasma (S. citri draft genome and all sequences longer that 500 nt), DNA viruses of arthropods (all complete genomes), all small RNA viruses of arthropods except dicistroviridae and iflavirus (complete genomes), all members of dicistroviridae and iflavirus except those infecting honey-bees (complete genomes), and all known honey bee RNA viruses (complete genomes). Each pool was converted into a Blast library and queried against the entire Solexa dataset by BlastN and tBlastx. Hits with an e-value greater than 161023 were extracted along with their paired end, regardless of similarity. Each pool was assembled using the Geneious sequence analysis package [102]. Contigs greater than 250 nt were queried again against the dataset by tBlastx with an e-value threshold of 161025. Any positive hits were then queried against the NR database with the same parameters to eliminate spurious hits. Contigs that appeared divergent or that were derived from non- honey bee associated viruses were extended using the entire read dataset using a paired-end contig extension algorithm (‘‘PRICE’’ Graham Ruby, manuscript under preparation, software available at http://derisilab.ucsf.edu). The extended contigs were then independently confirmed by PCR recovery and Sanger sequenc- ing. Individual paired-end reads that were discordant with the recovered contigs were used to further nucleate new contigs via contig extension. Primer3 [103] was used to design primers bridging adjacent contigs, as determined by mapping onto known virus genomes. Individual viruses or other microbes were queried with a BlastN threshold e-value of 161027 (W7) to determine read counts. Statistical Analysis Associations were calculated treating each hive sample at each time-point as a distinct event. P-values (Chi-square values) and PLoS ONE | www.plosone.org 15 June 2011 | Volume 6 | Issue 6 | e20656