and young bee samples as well as hive bottom boards. More significantly, monitor colonies received miticide treatments in order to reduce V. destructor burden. Deep sequencing analysis revealed the presence of four novel viruses (ALPV-Brookings, BSRV, LSV1 and LSV2), illustrating the power of deep short read sequencing and de novo assembly for virus discovery. Significantly, LSV2 was the most abundant single component of the honey bee microbiome in our study, and it is likely that the reason this virus has previous gone undetected is the fact that the Lake Sinai viruses are extremely divergent from all known insect viruses in both amino acid identity and genome organization. The non-structural genes of LSV are most closely related to CBPV, a known pathogen of honey bees [57], as well as other members of the Nodavirales. However, the capsid gene and monopartite genome structure resemble tetraviruses and together position this virus closer to species such as the Providence virus, which similarly has a monopartite genome, a Nodavirales-like polymerase and a tetravirus-like capsid. Since the presence of viral nucleic acid does not necessarily indicate infection, as pollen pellets of infected and non-infected workers are known to harbor honey bee viruses [88], we confirmed the presence of the replicative forms of LSV1 and 2 in time-course samples. The enormous magnitude of LSV throughout the time-course also suggests that these are bona fide honey bee viruses. LSV2 was the most abundant virus in our study and exhibited a unique seasonality. It is intriguing that peak LSV2 copy number per bee occurred in April (,3.661011) and January (,7.161011) since colonies typically collapse during the winter months. In contrast, LSV1 copy number peaked in July, similarly to the previously described honey bee viruses monitored in our study. Frequent detections of both ALPV-Brookings and BSRV (,15% incidence in the time-course) by PCR screen in different geographic regions argues against simple carryover from other insects during foraging, but does not rule out potential re-infection from stored pollen (bee bread) [88]. Research to determine the potential pathogenicity of these four new viruses in honey bees is underway. There are a number of previously identified honey bee viruses described on the basis of serology and electron microscopy (Bee virus X [95], Bee virus Y [96] Arkansas bee virus [97] and Berkeley bee virus [98] for which no nucleic acid sequence information is available in public databases. Without such data we cannot preclude the possibility that these previously described viruses overlap with our novel viruses, however we were not able to gain access to any nucleic acid or serological reagent to address the question directly. Regardless, the nucleic acid sequences of the viruses reported herein are attached to publically accessible records in the form of GenBank accessions (LSV1 - HQ871931, LSV2 -HQ888865, ALPV Strain Brookings - Q871932 and BSRV - JF423195-8), such that any future viral samples may be directly compared, or if historical samples can be found and analyzed, they too can be compared. Crithidia mellificae was readily detected throughout the time- course. In contrast to most other prevalent microbes and viruses, relative Crithidia levels peaked in the winter (January 2010). The effects of C. mellificae on the honey bee host remain relatively uncharacterized compared to those of C. bombi on bumble bee, which include reduced worker fitness and colony survival [66,99]. To date, there are only a few reports of C. mellificae infection of honey bees in the literature including early work describing the first isolation and culture of this organism in 1967 from Australian honey bees [67]. This work tested the effect of feeding C. mellificae to honey bees and demonstrated similar mortality rates in infected and uninfected bees [67]. More recently, similar trypanosomatid prevalence and loads were reported in CCD-affected colonies and Temporal Analysis of the Honey Bee Microbiome healthy controls [9,10]. Although current data suggest that C. mellificae does not dramatically affect colony health additional pathogenesis research in honey bees is warranted considering the detrimental effects of C. bombi on bumble bee colonies. The importance of honey bees in agriculture and the emergence of CCD underscores the need to monitor honey bee associated viruses and microbes in healthy colonies over time. The confinement of Spiroplasma infection to a two-month window demonstrates the value of time-course sampling as opposed to single-collection screens. The development of high throughput platforms, such as the APM, will facilitate monitoring of exogenous agents in order to better understand their effect on honey bee health and survival. Our discovery and genomic characterization of four new viruses will facilitate future monitor- ing. Temporal characterization of these and the other microbes described herein offers a more complete view of the possible microbe-microbe and microbe-environment interactions. Further studies examining any subtle or combinatorial effects of these novel microbes are required to understand their role in colony health. Increased analysis of prospectively collected samples is essential to address the hypothesis that either one or more viruses and/or microbes cause CCD. To our knowledge, this is the first U.S. honey bee pathogen monitoring study to report both comprehensive pathogen incidence and relative abundance of specific pathogens over time. Results from our molecular analysis pipeline (APM, PCR, qPCR, ultra deep sequencing) provide a basis for future epidemiologic studies aimed at determining the causes of CCD. Materials and Methods Collaborating commercial beekeeping operation information Twenty monitor hives were established in April 2009 by a large- scale (.72,000 hives), migratory commercial beekeeping opera- tion (Mississippi, California, and South Dakota, U.S.A.) that experienced CCD-losses in 2007/08. Standard beekeeping management practices for an operation of this size were employed. Treatment regimes throughout the year were as follows: (1) anti- mite treatment April 2009, just prior re-queening – amitraz; (2) antibacterial treatment May 2009 - oxytetracycline hydrochloride (OTC) (TerramycinTM); (3) anti-fungal (Nosema sp.) treatment August 25, September 12, and October 13, 2009 - fumagillan; (4) antibacterial treatment late August, early September, 2009 - tylosin tartrate; (5) anti-mite treatment September 12, 2009, after harvesting honey; (6) anti-mite treatment – early November and early December 2009 - essential oils from lemon grass and spearmint (Honey-B-HealthyTM). Honey bees colonies were periodically supplemented with sugar syrup and protein supple- ment. In April (1 gallon) and October (2 gallons) bees were fed 50% (weight/volume) sucrose; in November all colonies received 3 gallons of a 1:1 mixture of high fructose corn syrup-55 (HFCS-55, 55% fructose, 42% glucose) and sucrose syrup. Additional sugar syrup was given to colonies based on colony weight (,80 lbs - 3 gallons, 80–90 lbs - 2 gallons., 90–100 lbs – none). This operation experienced an average 18% colony loss from November 2009 to February 2010. Colonies with younger queens (#2 years old) experienced 11% loss, whereas colonies with older queens experience 21% loss. Honey Bee sampling and storage Samples (,50–100 bees) were collected into 50 mL Falcon tubes using a modified hand-held vacuum cleaner from both the entrance and interior of the hive and immediately put on dry ice PLoS ONE | www.plosone.org 12 June 2011 | Volume 6 | Issue 6 | e20656