Temporal Analysis of the Honey Bee Microbiome NC006265). Protein sequences were aligned by ClustalW and a tree generated by the Neighbor-Joining method with 100 replicates [102] (B) Genome organization of the Lake Sinai viruses and similar RNA viruses. doi:10.1371/journal.pone.0020656.g005 although the range A. borealis as a bumble bee pathogen extends across North America [74]. Discussion The importance of honey bees to global agriculture and the emergence of CCD calls for increased longitudinal monitoring of infectious processes within honey bee colonies. The data presented herein represent the finest resolution time-course of honey bee associated microbes to date. We demonstrate the utility of an arthropod pathogen microarray (APM) for simultaneous detection of numerous pathogens and the power of ultra deep sequencing for viral discovery. Several previous studies examined honey bee samples from diseased or CCD-affected and healthy colonies [9,10,20,75,76], but few have temporally monitored multiple pathogens [60,77,78]. Although these studies differed in sampling strategy, geography, colony management (e.g. migratory commer- cial versus stationary hobbyist, chemically treated versus organic), and pathogen monitoring technology (e.g. serology, PCR, spore counts, microarray) they provide a framework for our surveillance of previously characterized honey bee pathogens. Nosema infection was prevalent in our 20 monitor colonies. N. ceranae was the predominant species. N. apis was detected in individual colony samples in April (Mississippi) and May (South Figure 6. Crithidia mellificae, SF strain detection and quantification. (A) Light and fluorescent microscope images illustrate key features of this trypanosomatid parasite including DAPI stained kinetoplast DNA (yellow arrow) and nuclear DNA (white arrow), as well as the flagellar pocket (bottom panel, red arrow); scale bar = 5 mm. (B) Arthropod pathogen microarray detection of Crithidia mellificae in each colony (5 bees per sample) from October 2009 to January 2010. (C) Relative abundance of Crithidia mellificae throughout the time-course as assessed by RT-qPCR of pooled monthly time-course samples; quantification of rRNA copy number based on a standard curve as described in Materials and Methods. doi:10.1371/journal.pone.0020656.g006 PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e20656