Temporal Analysis of the Honey Bee Microbiome Figure 4. Relative abundance of select viruses assessed by RT-qPCR of pooled monthly time-course samples. Viral genome copy numbers per 100 ng RNA were calculated based on standard curves [(black queen cell virus (BQCV), sacbrood virus (SBV), acute bee paralysis virus (ABPV), Lake Sinai virus strain 1 (LSV1), Lake Sinai virus strain 2 (LSV2), aphid lethal paralysis virus strain Brookings (ALP-Br), and Big Sioux River virus (BSRV)]; multiplying reported values by 500 provides a copy number per bee estimate, as further described in Materials and Methods. LSV2, a novel virus, reached the highest copy number observed in this study in January 2010 (1.426109 copies per 100 ng of RNA sample; approximately 7.161011 copies per bee); note the y-axes on each graph are independently scaled. doi:10.1371/journal.pone.0020656.g004 enabled robust C. mellificae detection in later time-course samples, 33% of which tested positive (Figure 6). In addition, we screened samples throughout the time-course (April 2009–Jan. 2010) by PCR and qPCR specific to the C. mellificae rRNA gene. C. mellificae infection was detected by PCR at every time-point and in turn from every geographic location sampled in our study (MS, SD and CA). Likewise, C. mellificae was readily detected in pooled monthly RNA samples by qPCR throughout the year (Figure 6C). In contrast to BQCV, SBV, ABPV and Nosema ceranae, which exhibited peak levels in late summer and early fall, peak trypanosomatid levels occurred in January 2010. Despite this, C. mellificae infections statistically associated with N. ceranae infections (Chi Square p = 0.004, OR = 3.1). C. mellificae was also detected in numerous hobbyist and study hives in the San Francisco Bay Area (CA), as well as samples from a CCD-affected apiary in Oklahoma, indicating wide geographic distribution (Table S1). Spiroplasma melliferum and S. apis. Spiroplasma, a close relative of the genus Mycoplasma, are bacterial parasites that have been implicated as pathogens of insects, vertebrates and plants. Strains of spiroplasma similar to flower-associated parasites were identified as a pathogen of honey bees in France, Spiroplasma apis [69], and the United States, Spiroplasma melliferum [70]. Pilot Sanger sequencing of a pooled honey bee sample (August 2009) identified an rRNA-derived sequence from a Spiroplasma. Pan-spiroplasma and pan-mycoplasma PCRs targeting the 16S rRNA gene detected sporadic infections over most of the time-points and a surge of 9 infections in August and 6 infections in September [71]. Sequence data indicates that these isolates have high homology to previ- ously identified spiroplasma isolates (.98% nucleotide identity). Spiroplasma infections had strong associations with N. ceranae (Chi Square p = 0.015, OR = 7.2) and C. mellificae (p = 0.000076, OR = 16.3), however this may be an artifact of the short surge of Spiroplasma coinciding with a period of high Nosema load. Phorid fly (Apocephalus borealis). Apocephalus borealis, phorid flies, have previously been associated with bumble bee parasitism [72] and have recently been described as a parasite of honey bees in the San Francisco Bay Area [73]. Phoridae family members (e.g. Pseudacteon sp.) are well-characterized parasites of ants and other insects. These flies lay eggs inside the insect hosts, which are in turn consumed by the larvae during development. Although, A. borealis parasitism of honey bees is uncommon, we analyzed our time-course samples for the presence of phorid rRNA by PCR. Pooled monthly samples were weakly positive for Apocephalus borealis in December and January (Figure S3) and two individual hive samples produced robust amplicons. We sequenced PCR amplicons from two individual (September 2009 Hive #7 and October 2009 Hive #10) and one pooled-monthly (December 2009) samples and determined that the phorid rRNA sequences from our time-course shared 99% similarity to honey bee-parasitizing phorids captured in San Francisco. This is the first report of phorid flies in honey bee samples outside of California and thus expands their known geographic range (SD, CA), PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20656