Dakota), but was undetectable in pooled monthly samples, indicating relatively low levels. N. ceranae abundance peaked in early-spring and late-summer. Lower N. ceranae levels from November to January likely reflects antifungal (Fumagillan) treatments applied in the fall, but may also represent natural seasonal variation [58]. In comparison, another U.S.-based (Mississippi, Arkansas) study, which calculated Nosema levels using qPCR of genomic DNA calibrated to spore counts, also reported overall dominance by N. ceranae, but higher Nosema levels in November 2008 as compared to March 2009 [79]. Nosema spore count data from non-CCD and CCD-affected colonies in California and Florida was not significantly different and approximately 50% of the colonies assayed were infected [10]. Data from European studies indicate varying prevalence of N. apis and N. ceranae [25,29,80,81]. For example, a retrospective analysis of honey bee samples from Spain, Switzerland, France and Germany indicated peak levels of Nosema (presumably N. apis) in early spring and mid-winter from 1999 to 2002, whereas from 2003 to 2005 Nosema incidences remained relatively high throughout the year, a result the authors attribute to increased prominence of N. ceranae associated with recent increased bee losses [29]. In contrast, a recent (2005–2009) time-course study in Germany demonstrated greater Nosema incidence in the spring, detected N. apis more frequently than N. ceranae, and found no correlation between colony loss and Nosema infection [80]. Variable Nosema species prevalence and abundance at both the apiary and individual colony level indicate that standardized, molecular biology-based monitoring of large sample cohorts is required in order to understand the dynamics of Nosema infection, which are likely influenced by multiple factors including host genetic variation, climate, exposure levels, and treatment regimes [79,82]. Recently, higher levels of Nosema bombi were detected in North American bumble bee species experiencing population decline [83]. Although, like CCD, the causes of bumble bee decline are complex and not fully characterized, this report underscores the importance of further characterizing the epide- miology and pathogenicity of Nosema. We monitored the incidence of all known honey bee viruses, discovered 4 new honey bee associated viruses, and quantified the relative abundance of select viruses in time-course samples. Overall, no chronic infections of previously characterized honey bee viruses were observed and our data suggest that healthy colonies are undergoing constant cycles of viral infection. The most prevalent, previously characterized viruses in our study were BQCV, ABPV and SBV. The peak incidence of BQCV (25%) occurred in July, whereas ABPV (6.3%) and SBV (12.5%) peaked in August. Summer peak virus incidence was also reported in a PCR based honey bee virus (BQCV, ABPV, and SBV) survey of 36 geographically distributed apiaries in France (BQCV, ABPV, DWV, SBV, CBPV, KBV) [78], a qPCR time-course study of 15 colonies in England (BQCV and ABPV) [60], and an unpublished East-coast U.S. based survey (BQCV) [19]. Another virus, invertebrate iridescent virus-6, claimed to be associated with CCD and prevalent (75%) in healthy colonies but not supported in subsequent analysis [84,85], was never detected by the APM (n = 431), end-point PCR (n = 197), or in any of the 20 samples that were deep sequenced [86]. Seasonality of specific pathogens in our time-course study representing 2,155 individual bees from 431 samples varied, although many including BQCV, APBV, SBV, Nosema, exhibited reduced June and peak August levels. Peak incidences of these organisms in the spring and late summer are likely attributable to increased brood rearing [19,78,87] and foraging during these seasons [88]. Increased brood rearing during the summer, results Temporal Analysis of the Honey Bee Microbiome in a greater number of bees capable of transmitting pathogens to other members of the colony living in very close proximity [19]. Honey bee viruses are transmitted vertically via infected queens and horizontally via the oral-fecal route or through the exoskeleton [19,21]. Foraging activity also increases pathogen exposure [88] and may also stress the bees so that inapparent infections reach detectable levels. Although other sources of stress, such as transportation and poor nutrition, are hypothesized to increase pathogen levels [10], these factors were minimal during the summer when the monitor colonies were stably situated in South Dakota foraging on diverse pollen and nectar sources, including alfalfa (Medicago sativa L.), sweet clover (Melilotus spp.) and a variety of other flowering plants in June with increasing availability of corn (Zea mays ssp.) and soybean (Glycine max) pollen later in the summer. Notably, these colonies were part of a typically managed commercial beekeeping operation and there- fore received nutritional supplements, protein paddies and sugar syrup throughout the year (Materials and Methods). Adequate monitor colony nutrition may have played an important role in the rapid virus clearance observed in our study. Although further experimental validation is needed, recent work examining the effects of nutrition on DWV titer in caged-bee studies demon- strated that viral titer was reduced by pollen and protein supplementation [89]. In addition, anti-mite and antimicrobial treatments in the spring and late-fall may have accounted for the lower pathogen levels at those times of year and in turn for the relatively high levels during the summer (Materials and Methods). We did not observe either increased incidence or abundance of any of the microbes and viruses monitored in our study after long distance transport. Although several monitor colonies were lost (n = 3; one unfertile (drone laying) queen, two queen-less colonies) and many (n = 8) had fewer than 6 frames of bees in February 2010, none exhibited CCD characteristics and none of the numerous viruses and microbes we surveyed correlated with the weak colonies. Interestingly, our sample cohort had very few incidences of IAPV and DWV. IAPV, a virus that has received much attention due to its correlation with CCD-affected samples in an early study [9], although not in a subsequent expanded study [10], was detected in our monitor hives in December. The colonies in our study cleared or reduced IAPV infection to levels below detection within one week, indicative of a mild infection (Figure 3). IAPV infection has been shown to cause paralysis and death in mini-colony and cage studies [14,90], although its role in CCD is unclear [10,91,92]. Likewise, DWV incidence in our time-course samples was very low (0.7%) and presumably cleared rapidly. In contrast, a French time- course documented increased DWV incidence throughout the year (spring 56%, summer 66%, autumn 85%) [78] and two U.S. studies also report high DWV incidence [19,76]. Our results are not indicative of poor DWV detection by the array or our sampling strategy, since DWV was detected in both entrance and interior samples from other colonies. In addition, DWV-specific PCR of pooled monthly time-course samples was negative (Figure S3). Therefore negligible DWV in our monitor colonies may be attributed to low exposure and/or good colony health. A thorough one-year investigation of virus (ABPV, BQCV, DWV) and V. destructor in England found a correlation between DWV copy number and over-winter colony loss [60]. Lack of DWV in our monitor colonies is consistent with low Varroa destructor incidence, since mites are known to transmit DWV [39,93,94]. Low incidence of both DWV and V. destructor in our study may be partially attributed to our analysis of entrance samples, which consist of actively foraging and/or guarding adult bees. Since Varroa mites parasitize larva they are more readily detected in larva PLoS ONE | www.plosone.org 11 June 2011 | Volume 6 | Issue 6 | e20656