Temporal Analysis of the Honey Bee Microbiome Figure 2. Nosema detection and quantification in time-course samples from 20 honey bee colonies. (A) Arthropod pathogen microarray detection of Nosema sp. in each colony (5 bees per sample) throughout the 10-month time-course. Colonies were managed using standard commercial beekeeping practices and treatments, which are listed below panel A and further described in Materials and Methods. (B) Nosema ceranae and Nosema apis incidence assessed by species-specific end-point PCR from a single time-point (n = 20) each month; the positive sample percentages in each pie-chart are indicated in red. (C) Relative abundance of Nosema ceranae throughout the time-course assessed by qPCR of pooled monthly RNA samples; quantification of rRNA copy number based on a standard curve as described in Materials and Methods. doi:10.1371/journal.pone.0020656.g002 virus (LSV1) (HQ871931), after Lake Sinai in Brookings County, South Dakota. The second contig also represented a related, yet divergent (71% nt identity), RNA virus which we designated Lake Sinai virus 2 (LSV2) (HQ888865). The 59 end of LSV1 was determined by RACE (rapid amplification of cDNA ends). The 59 end of the LSV2 assembly was within 57 nt of the LSV1 RACE results [18,57]. The 39 ends of both viruses were refractory to traditional RACE methods and attempts at 59 RACE on the negative strand were also unsuccessful. Denaturing gel electrophoresis coupled with Northern bot analysis using three distinct LSV-specific probes indicated that only the monopartite genome and no subgenomic RNA species were present (Figure S4). Both LSV genomes display similarities to the RNA1 molecule of chronic bee paralysis virus (CBPV) with predicted open reading frames (ORFs) of similar size and arrangement with the notable exception that LSV1 and 2 ORFs are contained on a single RNA rather than in the bipartite configuration of CBPV [18,57] (Figures 5B and Figure S4). LSV1 and 2 possess the Orf1 gene, which is of unknown function, with predicted products (of 847 and 846 aa) previously unique to CBPV (853 aa). The Orf1 genes of LSV1 and CBPV share minimal (18%) amino acid identity. All three viruses encode an RdRp that partially overlaps and exists in a frame shift with respect to Orf1 [18]. Both LSVs possess a triple stop codon within 10 residues of the end of the Orf1 gene whereas CBPV has two adjacent stop codons. The RdRp genes are considerably more conserved with 80% identity between the two LSV strains and 25% amino acid identity between them and CBPV. Both LSV RdRp genes have the DxSRFD and SG amino acid motifs in the NTP binding pocket (residues 375–380 and 436– 437 in LSV1) conserved between the families Nodaviridae,Tombus- viridae and CBPV. An amino acid phylogeny of the Nodavirales superfamily RdRp places the LSV strains on the same branch as CBPV, and separated from the larger Nodavirus and Tombusvirus families (Figure 5A). As previously noted, the capsid protein of LSV1 and 2 is encoded on the same RNA as Orf1 and the RdRp unlike that of CBPV, which possesses a bipartite genome (Figure 5B). The capsids of LSV1 and 2 have significant profile similarity to the capsid gene of Nudaurelia capensis beta-tetravirus by HHpred [63] (e- value 1.0610226) and they exhibit weak direct protein alignment by Blastx (e-value 1.0610204). Similarity to tetravirus capsid genes consistently outranked similarity to CBPV or nodavirus capsids by these methods. Tetraviruses are not close relatives of the Nodavirales superfamily, although Betatetraviruses have a similar monopartite genome organization to LSV (Figure 5B). LSV1 and 2 share 70% amino acid identity over the capsid. The LSV1 capsid overlaps the RdRp gene in the +1 reading frame for 125 nt before ending in a pair of stop codons (separated by two residues). The LSV2 capsid is in frame with the RdRp and separated by 18 nt without a redundant stop codon. Seven of twenty hives sampled on August 5, 2009 were positive for LSV1 and an additional five hives in the time-course, from July (SD) and January/February (CA) were found to be positive for LSV1, all with greater than 95% nucleotide identity. LSV2 was more prevalent and was detected by PCR in 30 of 197 time-course samples from all three geographic regions. LSV2 incidence surged in April, July and January during which over a third of all 20 monitor hives were infected. Strain specific qPCR demonstrated high abundance ($26106 copies per 100 ng RNA) of both LSV strains in our monitor colonies throughout the majority of the time-course (Figure 4). LSV1 copy number peaked in July, at 1.396108 copies per 100 ng of RNA sample (approximately 7.061010 copies per bee). Notably, LSV2 was the most abundant virus detected in this study (,1011 copies per bee). Copy number peaked in both April and January, at 7.226108 copies per 100 ng of RNA sample (approximately 3.6161011 copies per bee) and 1.426109 copies per 100 ng of RNA sample (approximately 7.161011 copies per bee), respectively. Positive sense RNA viruses, like LSV 1 and 2, utilize a negative strand template to produce viral genome copies, therefore detection of the negative-strand intermediate is indicative of an actively replicating infectious virus [39,64,65]. We used negative- strand specific RT-PCR to detect the replicative forms of both LSV1 and LSV2 (Figure S5). cDNA synthesis reactions were performed using tagged negative strand-specific LSV1 and 2 primers followed by exonuclease I digestion of excess unincorpo- rated RT-primers [65] (Materials and Methods and Table S2). PCR amplification using a tag-specific forward primer and LSV- specific reverse primers confirmed the presence of the replicative forms of both LSV1 and LSV2 in the July RNA sample (Figure S5). Together, this data and the abundance of LSV1 and 2, compared to other significant honey bee viruses, suggests that LSV1 and LSV2 are novel honey bee viruses that may play significant roles in colony health. Crithidia mellificae. The broad scope of our microarray platform enabled identification of an unexpected microbe, Crithidia mellificae, in our time-course samples (Figure 6). Given that Crithidia bombi is a bumble bee pathogen and trypanosomatids were previously described in honey bees [9,66,67], 5 unique oligonucleotides each from Crithidia oncopelti and C. fasciculata rRNA sequences were included on the microarray. Oligonucleotides from these two distantly related organisms were predicted to hybridize to all other Crithidia species with published sequence [53]. Three oligonucleotides and their reverse complements derived from Crithidia oncopelti were repeatedly detected in samples throughout the time-course. Pilot Sanger sequencing of randomly amplified genomic DNA from a honey bee intestinal sample yielded a 121 base-pair (bp) stretch of the kinetoplast minicircle with 74% homology to the Crithidia fasciculata kinetoplast (BlastN e- value = 3.561028). Specific PCR retrieved 593 nt of the GAPDH gene to confirm phylogenetic placement [68]. We sought to further characterize this parasite by microscopy, PCR, culturing and DNA sequencing. Honey bee intestines were dissected in a sterile environment from which Crithidia mellificae was cultured. Light microscopy of these parasites enabled visualization of the flagella and motility (Figure 6; Figure S6 and Figure S7). Fixed sample imaging facilitated DAPI visualization of the kinetoplast DNA, as well as nuclear DNA (Figure 6). Previous studies describing trypanosomatids in honey bees lacked DNA- sequencing data with the exception of Cox-Foster et al. (2007) who published a 715-nt sequence of 18S ribosomal RNA that was too conserved between trypanosomatids for precise taxonomic assign- ment [9]. Together, the features observed by microscopy (flagella PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20656