Understanding the diversity and consequences of viruses present in honey bees is critical for maintaining pollinator health and managing the spread of disease. The viral landscape of honey bees ( Apis mellifera ) has changed dramatically since the emergence of the parasitic mite Varroa destructor , which increased the spread of virulent variants of viruses such as deformed wing virus. Previous genomic studies have focused on colonies suffering from infections by Varroa and virulent viruses, which could mask other viral species present in honey bees, resulting in a distorted view of viral diversity. To capture the viral diversity within colonies that are exposed to mites but do not suffer the ultimate consequences of the infestation, we examined populations of honey bees that have evolved naturally or have been selected for resistance to Varroa . This analysis revealed seven novel viruses isolated from honey bees sampled globally, including the first identification of negative-sense RNA viruses in honey bees. Notably, two rhabdoviruses were present in three geographically diverse locations and were also present in Varroa mites parasitizing the bees. To characterize the antiviral response, we performed deep sequencing of small RNA populations in honey bees and mites. This provided evidence of a Dicer-mediated immune response in honey bees, while the viral small RNA profile in Varroa mites was novel and distinct from the response observed in bees. Overall, we show that viral diversity in honey bee colonies is greater than previously thought, which encourages additional studies of the bee virome on a global scale and which may ultimately improve disease management. IMPORTANCE Honey bee populations have become increasingly susceptible to colony losses due to pathogenic viruses spread by parasitic Varroa mites. To date, 24 viruses have been described in honey bees, with most belonging to the order Picornavirales . Collapsing Varroa -infected colonies are often overwhelmed with high levels of picornaviruses. To examine the underlying viral diversity in honey bees, we employed viral metatranscriptomics analyses on three geographically diverse Varroa- resistant populations from Europe, Africa, and the Pacific. We describe seven novel viruses from a range of diverse viral families, including two viruses that are present in all three locations. In honey bees, small RNA sequences indicate that these viruses are processed by Dicer and the RNA interference pathway, whereas Varroa mites produce strikingly novel small RNA patterns. This work increases the number and diversity of known honey bee viruses and will ultimately contribute to improved disease management in our most important agricultural pollinator.

TheResistance to Dieldringene,Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized inDrosophila melanogasterby genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala³⁰¹to Ser. The equivalent change was subsequently identified inRdlorthologs of a large range of resistant insect species. Here, we report identification of a duplication at theRdllocus inD. melanogaster. The 113-kb duplication contains one WT copy ofRdland a second copy with two point mutations: an Ala³⁰¹to Ser resistance mutation and Met³⁶⁰to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser³⁰¹homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination betweenRootransposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kbRdllocus with a Ser³⁰¹change into an Ala³⁰¹background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of theRdltarget site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene.

Significance: Intergenerational epigenetic inheritance is usually considered to be of minor significance in mammals because the epigenome is reprogrammed twice between each generation. Yet whether this paradigm holds in nonmammalian organisms is largely unknown. Here, we show that epigenetic marks are transferred across generations in an invertebrate, the honey bee. We found that the epigenome is much more similar between fathers and daughters than between unrelated males and females of different generations within each colony. Epigenetic marks are not only conserved across generations, but also across somatic and germline tissues. Epigenetic reprogramming may thus be a mammalian-specific feature, suggesting a heightened capacity for epigenetic marks to influence evolutionary adaptation across the tree of life.

In honey bees (Apis mellifera), the epigenetic mark of DNA methylation is central to the developmental regulation of caste differentiation, but may also be involved in additional biological functions. In this study, we examine the whole genome methylation profiles of three stages of the haploid honey bee genome: unfertilised eggs, the adult drones that develop from these eggs and the sperm produced by these drones. These methylomes reveal distinct patterns of methylation. Eggs and sperm show 381 genes with significantly different CpG methylation patterns, with the vast majority being more methylated in eggs. Adult drones show greatly reduced levels of methylation across the genome when compared with both gamete samples. This suggests a dynamic cycle of methylation loss and gain through the development of the drone and during spermatogenesis. Although fluxes in methylation during embryogenesis may account for some of the differentially methylated sites, the distinct methylation patterns at some genes suggest parent-specific epigenetic marking in the gametes. Extensive germ line methylation of some genes possibly explains the lower-than-expected frequency of CpG sites in these genes. We discuss the potential developmental and evolutionary implications of methylation in eggs and sperm in this eusocial insect species.

The arrival of the ectoparasitic mite Varroa destructor on the western honeybee Apis mellifera saw a change in the diversity and prevalence of honeybee RNA viruses. One virus in particular, deformed wing virus (DWV) has become closely associated with V. destructor , leading many to conclude that V. destructor has affected viral virulence by changing the mode of transmission. While DWV is normally transmitted via feeding and faeces, V. destructor transmits viruses by direct injection. This change could have resulted in higher viral prevalence causing increased damage to the bees. Here we test the effect of a change in the mode of transmission on the composition and levels of honeybee RNA viruses in the absence of V. destructor . We find a rapid increase in levels of two viruses, sacbrood virus (SBV) and black queen cell virus (BQCV) after direct injection of viral extracts into honeybee pupae. In pupae injected with high levels of DWV extracted from symptomatic adult bees, DWV levels rapidly decline in the presence of SBV and BQCV. Further, we observe high mortality in honeybee pupae when injected with SBV and BQCV, whereas injecting pupae with high levels of DWV results in near 100% survival. Our results suggest a different explanation for the observed association between V. destructor and DWV. Instead of V. destructor causing an increase in DWV virulence, we hypothesize that direct virus inoculation, such as that mediated by a vector, quickly eliminates the most virulent honeybee viruses resulting in an association with less virulent viruses such as DWV.

The epigenetic mark of DNA methylation, the addition of a methyl (CH₃) group to a cytosine residue, has been extensively studied in many mammalian genomes and, although it is commonly found at the promoter regions of genes, it is also involved in a number of different biological functions. In other complex animals, such as social insects, DNA methylation has been determined to be involved in caste differentiation and to occur primarily in gene bodies. The role of methylation in nonsocial insects, however, has not yet been explored thoroughly. Here, we present the whole-genome DNA methylation profile of the nonsocial hymenopteran, the jewel wasp (Nasonia vitripennis). From high-throughput sequencing of bisulfite-converted gDNA extracted from male Nasonia thoraces, we were able to determine which cytosine residues are methylated in the entire genome. We found that an overwhelming majority of methylated sites (99.7%) occur at cytosines followed by a guanine in the 3′ direction (CpG sites). Additionally, we found that a majority of methylation in Nasonia occurs within exonic regions of the genome (more than 62%). Overall, methylation is sparse in Nasonia, occurring only at 0.18% of all sites and at 0.63% of CpGs. Our analysis of the Nasonia methylome revealed that in contrast to the methylation profile typically seen in mammals, methylation is sparse and is constrained primarily to exons. This methylation profile is more similar to that of the social hymenopteran species, the honey bee (Apis mellifera). In presenting the Nasonia methylome, we hope to promote future investigation of the regulatory function of DNA methylation in both social and nonsocial hymenoptera.