Israel acute paralysis virus (IAPV) is associated with colony collapse disorder of honey bees. Nonetheless, its role in the pathogenesis of the disorder and its geographic distribution are unclear. Here, we report phylogenetic analysis of IAPV obtained from bees in the United States, Canada, Australia, and Israel and the establishment of diagnostic real-time PCR assays for IAPV detection. Our data indicate the existence of at least three distinct IAPV lineages, two of them circulating in the United States. Analysis of representatives from each proposed lineage suggested the possibility of recombination events and revealed differences in coding sequences that may have implications for virulence.Over the winter of 2006 and 2007, an estimated 23% of all beekeeping operations in the United States experienced losses of hives attributed to colony collapse disorder (CCD) (30). More than 90 cultivated crops depend on the honey bee, Apis mellifera, for pollination; thus, CCD has profound implications for the food supply (23). We recently reported that the presence in hives of the dicistrovirus Israel acute paralysis virus (IAPV) was strongly correlated with the presence of CCD (8).IAPV was first described in 2004 in Israel (21), where infected bees presented with shivering wings, progressed to paralysis, and then died outside the hive. IAPV has features comparable to those of members of the family Dicistroviridae of the superfamily Picornaviridae. Unlike members of the Picornaviridae, which have a single open reading frame (ORF) encoding a single polyprotein, viruses in the family Dicistroviridae have two ORFs encoding two polyproteins. Dicistroviruses have two internal ribosomal entry sites (IRES), one found in the 5Ј untranslated region (UTR) and the other located in the intergenic region between ORF1 and ORF2. Other viruses known to infect honey bees, and related to picornaviruses, are sacbrood virus, deformed wing virus, acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and black queen cell virus (4, 7).Given the importance of honey bees as pollinators and the reported association between CCD and IAPV, we pursued phylogenetic analysis of geographically discrete IAPV isolates. We report the complete genome sequences of representatives from each of three lineages of IAPV and demonstrate through detailed analysis of four regions of the viral genome (the 5Ј UTR and 5Ј terminus of ORF1; the 3Ј terminus of ORF1, the intergenic region, and the 5Ј terminus of ORF2; ORF2; and the RNA-dependent RNA polymerase [RdRp]) the presence on three continents of at least three clusters of IAPV, two of them currently circulating in the United States. MATERIALS AND METHODSSample collection and RNA extraction. Adult bees were collected from migratory beekeeping operations in the United States with CCD. Diseased apiaries were identified based on evidence of recent collapse of colonies within the apiary and a lack of dead bees in the collapsed colonies. From each colony, 150 adult bees were collected and stored at Ϫ80°C until they w...

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31Colony Collapse Disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey 32 bees, is thought to be responsible for a minority of the large over-wintering losses experienced by U.S. 33 beekeepers since the winter of 2006-2007. Using the same data set developed to perform a mono-34 factorial analysis (vanEngelsdorp et al. 2009), we conducted a classification and regression tree (CART) 35 analysis in an attempt to better understand the relative importance and inter-relations among different risk 36 variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: 37 one with and one without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The 38 resulting model tree which permitted for misclassification had a sensitivity and specificity of 85% and 39 59% respectively. While factors measuring colony stress (e.g., adult bee physiological measures such as 40 fluctuating asymmetry or mass of head, and morphological measures such as frames of brood) were 41important discriminating values, 6 of the 19 variables having the greatest discriminatory value were 42 pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly 43 used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. 44Our CART analysis provides evidence that CCD is likely the result of several factors acting in concert, 45 making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant 46 further attention, including the effect of sub-lethal pesticide exposure on pathogen prevalence and the role 47 of variability in bee tolerance to pesticides on colony survivorship. 48 49

Honey bee (Apis mellifera) queens mate with unusually high numbers of males (average of approximately 12 drones), although there is much variation among queens. One main consequence of such extreme polyandry is an increased diversity of worker genotypes within a colony, which has been shown empirically to confer significant adaptive advantages that result in higher colony productivity and survival. Moreover, honey bees are the primary insect pollinators used in modern commercial production agriculture, and their populations have been in decline worldwide. Here, we compare the mating frequencies of queens, and therefore, intracolony genetic diversity, in three commercial beekeeping operations to determine how they correlate with various measures of colony health and productivity, particularly the likelihood of queen supersedure and colony survival in functional, intensively managed beehives. We found the average effective paternity frequency (m e ) of this population of honey bee queens to be 13.6 ± 6.76, which was not significantly different between colonies that superseded their queen and those that did not. However, colonies that were less genetically diverse (headed by queens with m e  ≤ 7.0) were 2.86 times more likely to die by the end of the study when compared to colonies that were more genetically diverse (headed by queens with m e > 7.0). The stark contrast in colony survival based on increased genetic diversity suggests that there are important tangible benefits of increased queen mating number in managed honey bees, although the exact mechanism(s) that govern these benefits have not been fully elucidated.