Widespread distribution of honey bee-associated pathogens in native bees and wasps: Trends in pathogen prevalence and co-occurrence.

Pollinators have experienced significant declines in the past decade, in part due to emerging infectious diseases. Historically, studies have primarily focused on pathogens in the Western honey bee, Apis mellifera. However, recent work has demonstrated that these pathogens are shared by other pollinators and can negatively affect their health. Here, we surveyed honey bees and 15 native bee and wasp species for 13 pathogens traditionally associated with honey bees. The native bee and wasp species included 11 species not previously screened for pathogens. We found at least one honey bee-associated pathogen in 53% of native bee and wasp samples. The most widely distributed and commonly detected pathogens were the microsporidian Nosema ceranae, the bacterium Melissococcus plutonius, and the viruses deformed wing virus and black queen cell virus. The prevalence of viruses was generally higher in honey bees than in native bees and wasps. However, the prevalence of M. plutonius and the brood fungus Ascosphaera apis was significantly higher in some native bee species than in honey bees. The data also reveal novel trends in the association between co-occurring pathogens in honey bees and native bees and wasps at the pathogen community level. These results can inform the assessment of risks that native pollinator species face from pathogen stress, and indicate that many non-viral pathogens, notably M. plutonius and N. ceranae, are far more widely distributed and commonly found in native bees and wasps than previously thought.

In vitro infection of pupae with Israeli acute paralysis virus suggests disturbance of transcriptional homeostasis in honey bees (Apis mellifera).

The ongoing decline of honey bee health worldwide is a serious economic and ecological concern. One major contributor to the decline are pathogens, including several honey bee viruses. However, information is limited on the biology of bee viruses and molecular interactions with their hosts. An experimental protocol to test these systems was developed, using injections of Israeli Acute Paralysis Virus (IAPV) into honey bee pupae reared ex-situ under laboratory conditions. The infected pupae developed pronounced but variable patterns of disease. Symptoms varied from complete cessation of development with no visual evidence of disease to rapid darkening of a part or the entire body. Considerable differences in IAPV titer dynamics were observed, suggesting significant variation in resistance to IAPV among and possibly within honey bee colonies. Thus, selective breeding for virus resistance should be possible. Gene expression analyses of three separate experiments suggest IAPV disruption of transcriptional homeostasis of several fundamental cellular functions, including an up-regulation of the ribosomal biogenesis pathway. These results provide first insights into the mechanisms of IAPV pathogenicity. They mirror a transcriptional survey of honey bees afflicted with Colony Collapse Disorder and thus support the hypothesis that viruses play a critical role in declining honey bee health.

Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity.

We have identified 44 serine protease (SP) and 13 serine protease homolog (SPH) genes in the genome of Apis mellifera. Most of these genes encode putative secreted proteins, but four SPs and three SPHs may associate with the plasma membrane via a transmembrane region. Clip domains represent the most abundant non-catalytic structural units in these SP-like proteins -12 SPs and six SPHs contain at least one clip domain. Some of the family members contain other modules for protein-protein interactions, including disulphide-stabilized structures (LDL(r)A, SRCR, frizzled, kringle, Sushi, Wonton and Pan/apple), carbohydrate-recognition domains (C-type lectin and chitin-binding), and other modules (such as zinc finger, CUB, coiled coil and Sina). Comparison of the sequences with those from Drosophila led to a proposed SP pathway for establishing the dorsoventral axis of honey bee embryos. Multiple sequence alignments revealed evolutionary relationships of honey bee SPs and SPHs with those in Drosophila melanogaster, Anopheles gambiae, and Manduca sexta. We identified homologs of D. melanogaster persephone, M. sexta HP14, PAP-1 and SPH-1. A. mellifera genome includes at least five genes for potential SP inhibitors (serpin-1 through -5) and three genes of SP putative substrates (prophenoloxidase, spätzle-1 and spätzle-2). Quantitative RT-PCR analyses showed an elevation in the mRNA levels of SP2, SP3, SP9, SP10, SPH41, SPH42, SP49, serpin-2, serpin-4, serpin-5, and spätzle-2 in adults after a microbial challenge. The SP41 and SP6 transcripts significantly increased after an injection of Paenibacillus larva, but there was no such increase after injection of saline or Escherichia coli. mRNA levels of most SPs and serpins significantly increased by 48 h after the pathogen infection in 1st instar larvae. On the contrary, SP1, SP3, SP19 and serpin-5 transcript levels reduced. These results, taken together, provide a framework for designing experimental studies of the roles of SPs and related proteins in embryonic development and immune responses of A. mellifera.

Bacterial probiotics induce an immune response in the honey bee (Hymenoptera: Apidae).

To explore immune system activation in the honey bee, Apis mellifera L., larvae of four ages were exposed through feeding to spores of a natural pathogen, Paenibacillus larvae larvae, to cells of a diverse set of related nonpathogenic bacteria, and to bacterial coat components. These larvae were then assayed for RNA levels of genes encoding two antibacterial peptides, abaecin and defensin. Larvae exposed to either P. l. larvae or a mix of nonpathogenic bacteria showed high RNA levels for the abaecin gene relative to controls. First instars responded significantly to the presence of the nonpathogenic mix within 12 h after exposure, a time when they remain highly susceptible to bacterial invasion. This response was sustained for two successive instars, eventually becoming 21-fold higher in larvae exposed to probiotic spores versus control larvae. The mixture of nonpathogenic bacteria is therefore presented as a potential surrogate for assaying the immune responses of different honey bee lineages. It also is proposed that nonpathogenic bacteria can be used as a probiotic to enhance honey bee immunity, helping bee larvae, and other life stages, survive attacks from pathogens in the field.

Complete mitochondrial DNA sequence of the important honey bee pest, Varroa destructor (Acari: Varroidae).

Mites in the genus Varroa are the primary parasites of honey bees on several continents. Genetic analyses based on Varroa mitochondrial DNA have played a central role in establishing Varroa taxonomy and dispersal. Here we present the complete mitochondrial sequence of the important honey bee pest Varroa destructor. This species has a relatively compact mitochondrial genome (15,218 bp). The order of genes encoding proteins is identical to that of most arthropods. Ten of 22 transfer RNAs are in different locations relative to hard ticks, and the 12S ribosomal RNA subunit is inverted and separated from the 16S rRNA by a novel non-coding region, a trait not yet seen in other arthropods. We describe a dispersed set of 45 oligonucleotide primers that can be used to address genetic questions in Varroa. A subset of these primers should be useful for taxonomic and phylogenetic studies in other mites and ticks.