odds ratios (OR) listed were calculated by the OpenEpi statistical package v2.3 (http://www.openepi.com/OE2.3/Menu/OpenE- piMenu.htm). Only seven microbes with incidences in the study set of at least 10% (20 incidences in 197 samples) were examined for association, resulting in 28 discrete association tests and the corresponding Bonferroni multiple testing correction. Microbes occurring infrequently were not used in association tests and so did not contribute to multiple testing correction. Data Availability and Compliance with Standards APM design and results have been submitted to GEO (design accession GPL11490 and array data accession GSE28235) and are MIAME compliant. All Sanger sequence-confirmed deep se- quencing assemblies have been submitted to GenBank (accessions listed in text). Supporting Information Figure S1 Gel electrophoresis of RT-qPCR products from pooled-monthly samples. qPCR products were ampli- fied using the primer sets listed in Table S2: Nosema ceranae 249 bp, Crithidia mellificae 153 bp, black queen cell virus (BQCV) 141 bp, sacbrood virus (SBV) 103 bp, acute bee paralysis virus (ABPV) 177 bp, Lake Sinai Virus strain 1 (LSV1) 153 bp, Lake Sinai Virus strain 2 (LSV2) 225 bp, Aphid Lethal Paralysis Virus Strain Brookings (ALP-Br) 141 bp, and Big Sioux River virus (BSRV) 281 bp. Molecular weight ladder (L), April 2009 (A), May (M), June (J6), July (J7), August (A), September (S), October (O), November (N), December (D), January 2010 (J1); RNA no RT control (2), plasmid standard copy number (10X). (TIFF) Figure S2 Dicistrovirus Phylogeny. Dicistrovirus IRES elements were aligned by ClustalW and a Neighbor-Joining tree generated by the Geneious Tree Builder (100 replicates). IAPV – Israeli acute paralysis virus (NC009025), KBV – Kashmir bee virus (NC004807), ABPV – acute bee paralysis virus (NC002548), SINV1 – Solenopsis invicta virus 1 (NC006559), TSV – Taura syndrome virus (NC003005), ALPV – acute lethal paralysis virus (NC004365), ALPV strain Brookings (Q871932), RhPV – Rhopalosiphum padi virus (NC001874), BSRV – Big Sioux River virus (JF423195-8), CrPV – cricket paralysis virus (NC003924), DCV – Drosophila C virus (NC001834), TV – Triatoma virus (NC003783), HPV – Himetobi P virus (NC003782), PSV – Plautia Stali intestine virus (NC003779), HCV – Homalodisca coagulata virus (NC008029), and BQCV – black queen cell virus (NC003784); red text – common honey bee viruses; blue text – novel viruses. (TIFF) Figure S3 RT-PCR results from pooled-monthly sam- ples. (A) Nosema apis (268 bp), (B) deformed wing virus (DWV; 194 bp), (C) Apocephalus borealis (phorid fly; 500 bp), (D) Apis mellifera ribosomal protein L8 (Rpl8; 100 bp). Molecular weight ladder (L), April 2009 (A), May (M), June (J6), July (J7), August (A), September (S), October (O), November (N), December (D), References 1. Pennisi E (2006) Genetics - Honey bee genome illuminates insect evolution and social behavior. Science 314: 578–579. 2. Winston ML (1987) The biology of the Honey Bee: First Harvard Press. 3. Johnson R (2010) Honey Bee Colony Collapse Disorder. 4. USDA-ARS Questions and Answers: Colony Collapse Disorder. 5. Morse RA, Calderone NW (2000) The Value of Honey Bees as Pollinators of U.S. Crops in 2000. Gleanings in Bee Culture Supple. pp 1–15. 6. vanEngelsdorp D, Underwood R, Caron D, Hayes J (2007) An estimate of managed colony losses in the winter of 2006–2007: A report commissioned by the apiary inspectors of America. Am Bee J 147: 599–603. Temporal Analysis of the Honey Bee Microbiome January 2010 (J1); RNA only no RT control (2), water (H2O), and positive control (+). (TIFF) Figure S4 Detection of the LSV genome by denaturing 1.5% agarose gel electrophoresis and Northern blots using three LSV-specific probes. RNA (15 mg) extracted from the supernatants of homogenized honey bees was transferred to a membrane and probed using LSV-specific probes corre- sponding to different regions of the genome (P1 – 1482–1744, P2 – 2289–2477, and P3 – 4509–4714) as described in Materials and Methods. (TIFF) Figure S5 Detection of the replicative form of LSV1 and LSV2 by negative strand-specific RT-PCR. The pooled July RNA sample was analyzed for the presence of LSV negative- strand RNA, which is indicative of virus replication, using strand- specific RT-PCR as described in Materials and Methods; RT- PCR products from reactions were analyzed by agarose (2%) gel electrophoresis. (TIF) Figure S6 Crithidia mellificae, strain SF movies. Light microscopy of live parasites was performed using a Leica DM6000 microscope (1006 objective) equipped with Hamamatsu C4742- 95 camera and Volocity Software (PerkinElmer). (MP4) Figure S7 Crithidia mellificae, strain SF movies. Light microscopy of live parasites was performed using a Leica DM6000 microscope (1006 objective) equipped with Hamamatsu C4742- 95 camera and Volocity Software (PerkinElmer). (MP4) Arthropod pathogen microarray results from test Table S1 samples. (DOCX) Table S2 Primers used in this study, * denotes primer sets used for PCR screening results in Figure 3B, ** denotes qPCR primer sets used to obtain the results in Figure 4 and Figure S3. (DOCX) Acknowledgments We would like to thank Bret Adee and the staff at Adee Honey Farms, Inc., and Steve Thomas, San Francisco Beekeepers Association, for assistance with sampling; Christi Heintz, Executive Director Project Apis m., for facilitating this study; Christopher Smith and John Hafernik for sharing their phorid discovery results; and members of the DeRisi and Andino laboratories for reviewing this manuscript. Author Contributions Conceived and designed the experiments: JLDR MLF CR. Performed the experiments: CR MLF JCE. Analyzed the data: CR MLF JGR JLDR. Contributed reagents/materials/analysis tools: JGR DG RA JLDR. Wrote the paper: MLF CR RA DG JLDR. 7. vanEngelsdorp D, Hayes J, Caron D, Pettis J (2010) Preliminary Results: Honey Bee Colonies Losses in the U.S., winter 2009–2010. Apiary Inspectors of America (AIA) and USDA-ARS Beltsville Honey Bee Lab. 8. vanEngelsdorp D, Hayes J, Underwood RM, Pettis J (2008) A Survey of Honey Bee Colony Losses in the US, Fall 2007 to Spring 2008. PLoS ONE 3: e4071. 9. Cox-Foster DL, Conlan S, Holmes EC, Palacios G, Evans JD, et al. (2007) A metagenomic survey of microbes in honey bee colony collapse disorder. Science 318: 283–287. 10. vanEngelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E, et al. (2009) Colony collapse disorder: a descriptive study. PLoS ONE 4: e6481. PLoS ONE | www.plosone.org 16 June 2011 | Volume 6 | Issue 6 | e20656