Molecular Detection, Prevalence And Transmission Of Israeli Acute Paralysis Virus And Its Effects On Honey Bee Behaviors

The honey bee colony losses across parts of the world in recent years and that honey bee colonies disappeared abruptly in the US and parts of Europe from winter2006to spring2007have triggered pollination crisis. The sudden collapse of honeybee colonies is known as colony collapse disorder (CCD) and was initially thought to be caused by Israeli acute paralysis virus (IAPV). Following more research, it is generally accepted that a variety of factors including Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition may cause CCD. Besides, interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years.Viral prevalence of common honey bee viruses including IAPV, Deformed wing virus (DWV), Chronic bee paralysis virus (CBPV), Acute bee paralysis virus (ABPV), Sacbrood virus (SBV) and Kashmir bee virus (KBV) was surveyed in parts of Zhejiang province, and comparisons between Apis mellifera and Apis cerana for pathogen diversity and differential expression of immune genes were furthered analyzed in the study; first detection of honey bee viruses including IAPV, Black queen cell virus (BQCV) and DWV and their replication in the fungi Ascosphaera apis (A. apis) uncovers a new biological feature of honey bee viruses; effects of IAPV on sucrose responsiveness and homing ability of pollen foragers were investigated based on proboscis extension response (PER) and radio frequency identification (RFID) techniques; synergistic effects of two pathogens including Varroa mites and Nosema ceranae on IAPV titer dynamics and expression of immune genes in honey bees were also studied. These results were summarized as follows:1. A survey of six honey bee viruses in Zhejiang province and comparisons between Apis mellifera and Apis cerana for pathogen diversity and molecular mechanisms underlying the differential response to pathogensSamples collected from38typical apiaries located in7different regions of Zhejiang province were tested for presence of six common honey bee viruses. The most prevalent virus was IAPV, with the detection rate of29%in all samples, followed by DWV (27%), KBV (5%), SBV (5%), CBPV (4%) and ABPV (3%). Examination of tissue tropism of the viruses showed that thorax and abdomen of crawling bees and dead bees were more likely to be positive for viruses.A regional survey covering honey bee viruses, mite and wax moth infestation, pesticide poisoning, foulbrood, chalkbrood, which are detrimental to the health of colonies was carried out in the study. Of141apiary locations consisting of colonies of Apis mellifera ligustica (Aml),5%of apiaries suffered from inexplicable colony losses. Honey bee colonies from about80%of apiaries were observed with different levels of Varroa mite infestation.21%of apiaries had chalkbrood, and6%of apiaries had foulbrood.18%of apiaries underwent colony losses resulting from pesticide poisoning.39apiaries raising A. cerana cerana (Acc) did not confront with colony mortality or Varroa mites compared with A. mellifera. Nearly90%of Acc apiaries were more or less infected with the greater wax moth (Galleria mellonella), followed by foulbrood positive apiaries (18%).Given the differences in biology and pathogen diversity between Acc and Aml, we first used the Illumina-Solexa deep sequencing technology to describe the differences in the heads of Acc and Aml foragers at the gene expression level, and found about2,370differentially expressed genes related to metabolism, immune and stress response between Acc and Aml. The differential expression of antimicrobial peptide genes suggested that the defense mechanisms against pathogenic microbes in Acc and Aml are different from each other. That may be a reason why there were no reports about the unusual mortality of eastern honeybee (Acc) colonies in comparison to the drastic loss of western honeybee (A. mellifera) that has been occurring worldwide in recent years.2. First detection of honey bee viruses and their replication in the fungi Ascosphaera apisThe Ascosphaera apis (A. apis) is a fungal pathogen of honey bees causing chalkbrood in honey bee larvae and it has been shown that fungal viruses or mycoviruses have been found to be widespread in all major fungal taxa. Therefore, the chalkbrood mummies from which the A. apis was cultured and isolated, and the mycelium of A. apis were further used for detecting the common honey bee viruses in the present study. We demonstrated that honey bee viruses including DWV, Black queen cell virus (BQCV) and IAPV could infect and replicate in the fungal pathogen A. apis that causes honey bee chalkbrood disease, revealing a novel biological feature of honey bee viruses. Replication of honey bee viruses in A. apis indicated that A. apis may act as a possible vector during honey bee viruses transmission.The phylogenetic analysis showed that phylogenetic relationships between viruses of fungal and honey bee origins are all monophyletic, forming two clusters in the phylogenetic trees distinctly. Viruses from A. apis constitute a distinctive lineage, separated from the clades of viruses identified in honey bees. Further studies are warranted to investigate the impact of the viruses on the fitness of their fungal host and phenotypic effects the virus-fungus combination has on honey bee hosts.3. Viral infection affects sucrose responsiveness and homing ability of forager honey bees, Apis mellifera L.In the present study, the effects of a honey bee virus, IAPV, on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on PER assays and RFID systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around107copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. However, sensitization test and habituation test, two patterns of nonassociative learning, showed no significant difference between foragers infected with IAPV and foragers infected with PBS. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.4. Effects of Varroa mites and Nosema ceranae on IAPV titer dynamics and expression of immune genes in honey beesIAPV titer dynamics and expression of Abaecin, Eater and Vitellogenin (Vg) in honey bees can be affected by co-infection with Varroa mites and Nosema ceranae. However, no obvious trend regarding expression of immune genes was found in the study. Some immune genes showed trends opposite the expect trends. This preliminary study indicated that the complicated interactions beween Nosema ceranae、mites、IAPV and honey bees. Further study is needed regarding the complicated interactions.