Table 1. Cont. Acarapis Nematodes Positive Controls 2 30 Total: 32 doi:10.1371/journal.pone.0020656.t001 responsible for infections, each hive was analyzed at a single time- point per month by species specific PCR. In April and May, N.apis was predominant whereas in June, July, and October through December, N. ceranae was exclusively detected (Figure 2B). During the highest incidence of Nosema (August–September), 75% of all colonies were infected with Nosema ceranae and less than 25% with Nosema apis, most of which were co-infected with N. ceranae. Quantitative-PCR data from pooled monthly samples confirmed that Nosema ceranae was prevalent throughout the time-course and peaked in August (Figure 2C). While seasonal variation may play a role, an anti-fungal (Fumagillan) was used to abrogate Nosema infection [58] and may be responsible for the observed decrease in Nosema abundance from November to January (Figure 2). Viruses. The APM readily detected common honey bee viruses in samples collected throughout the time-course. In total, we report 69 virus incidences in 63 of 431 total samples (Figure 3). Overall virus incidence was sporadic, which we attribute to cycles of acute infection in predominantly healthy monitor colonies. The majority of infections occurred during July, August, and September when the monitor colonies were in South Dakota. The most prevalent virus infections observed during our 10-month study were SBV, BQCV and ABPV; however the frequencies of specific viruses were insufficient for statistical tests regarding pathogen association (see Materials and Methods). Other viruses including DWV, IAPV, and KBV were infrequently detected in the latter half of our time-course. A total of six double virus infections were observed, frequently involving ABPV or SBV. There were only two cases in which the same virus (BQCV) was detected in consecutive time points from a particular monitor colony (Figure 3A Hives #4 and #6). Typically a single virus was detected in multiple colonies at a given time-point and these infections did not persist. For example, there were waves of SBV infection in April and January and of BQCV in July and early August (Figure 3A). qPCR analysis of pooled monthly samples confirmed and extended APM findings. BQCV, SBV and ABPV levels peaked in mid-summer to early fall at 6.66109–861010 genome copies per bee (Figure 4 and Figure S1), consistent with previously characterized levels of these viruses [57,59,60]. Ultra deep sequencing, discovery of novel viruses. A summer South Dakota time-point (August 5, 2009) was selected for deep sequencing due to high Nosema load and the presence of several common honey bee viruses, including ABPV, BQCV and SBV. All expected microbes (Nosema ceranae, Crithidia mellificae) and viruses were detected [ABPV (39,352 reads aligned by BlastN e- value,161027), BQCV (2,868 reads) and SBV (4,414 reads)]. In addition, we detected Spiroplasma sequences (70,407 reads) consistent with the presence of both Spiroplasma apis and S. melliferum (66 reads and 44 reads aligning to the RNA PolB gene of each, respectively). Four distinct novel viruses were discovered via deep sequencing. Paired-end sequencing reads (2663 nt) of unknown origin were screened by tBlastx [61] against all known insect viruses present in GenBank [62]. Screening hits with an e-value greater than 161023 were used to target de novo contig assembly using the Temporal Analysis of the Honey Bee Microbiome complete data set. Short contigs were screened by tBlastx against the non-redundant nucleotide database (NR) at an e-value threshold of 161025. Hits to viral sequence, but not host sequences, were further assembled (see Materials and Methods). In each case, PCR primers were initially designed to bridge or confirm assembled contigs by Sanger sequencing. Confirmed contigs were extended with the PRICE assembler package (see Materials and Methods). In total, sequences from four novel viruses were recovered and Sanger validated. These include two members of Dicistroviridae, and two RNA viruses distantly related to Nodaviridae. Aphid Lethal Paralysis virus strain Brookings. Investigation of contigs aligning to the Aphid Lethal Paralysis Virus genome, in the family Dicistroviridae, recovered a 4,125 nt contig (GenBank Q871932) spanning the RNA-dependent RNA Polymerase (RdRp) gene, the internal ribosome entry site (IRES) structure and the capsid coding region. The recovered sequence aligned with 83% nucleotide and 89% amino acid identity to the canonical ALPV genome over the RdRp gene. The two viruses shared 97% nucleotide homology along 171 nt of the IRES. The high sequence similarity between this new isolate and canonical ALPV makes it unclear whether this is a novel species or a new strain of ALPV. Regardless, ALPV has not previously been reported in association with honey bees. We propose the designation ALPV strain Brookings (after the SD county from which the virus was isolated). Specific PCR primers were designed for the Brookings strain and used to analyze additional time-course samples, resulting in detections on thirty distinct occasions, including in Mississippi, South Dakota and California. Incidence peaked in May, when 7 out of 20 hives were infected, whereas maximum abundance occurred in August albeit at a relatively low level, 4.426104 copies per 100 ng of RNA sample (approximately 2.216107 copies per bee), as compared to previously characterized honey bee viruses (Figures 3 and 4). Frequent detection of ALPV strain Brookings throughout the time-course from multiple geographic locations suggests that this virus is not simply a ‘‘passenger’’ obtained from forage (nectar and pollen) shared with other insects. However, further investigation is required to determine whether ALPV strain Brookings is a honey bee pathogen. Big Sioux River virus. A second novel dicistrovirus, designated Big Sioux River Virus (BSRV) after its place of discovery, is most similar to the Rhopalosiphum padi Virus (RhPV). Four contigs of size 1473, 861, 1164 and 1311 nt (GenBank JF423195-8) derived from the non-structural region, the IRES, and the capsid gene. BSRV shares low amino acid identity with RhPV; only 78% in the non-structural region and 69% in the capsid gene. This level of amino acid divergence is consistent with the taxonomic rank of a new species (Figure S2). Twenty-eight incidences of BSRV were detected from 197 time-course samples by specific PCR with most individual colony detections occurring in samples collected from April to July 2009 in Mississippi and South Dakota. Incidence was low from October onwards (Figure 3B). Peak abundance was 7.646103 copies per 100 ng of RNA sample (approximately 3.86106 copies per bee) and occurred in August (Figure 4). Of note, BSRV associated significantly with Nosema apis infections (p = 0.003, OR 6.0) and also with ALPV-Brookings (p = 0.014, odds ratio (OR) = 4.5). Lake Sinai Virus strain 1 and 2. Three contigs had significant alignment to chronic bee paralysis virus (CBPV) and members of the family Nodaviridae. Both the individual reads and our initial contigs were further assembled and extended using the complete data set (see Materials and Methods). Two separate contig sequences (,5.5 kb each) were generated by de novo assembly. Both contigs were confirmed by specific PCR and Sanger sequencing. The first contig represents a novel RNA virus that we designate Lake Sinai PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20656