Katherine A. Aronstein

(2015). A multiplex PCR assay for determination of mating type in isolates of the honey bee fungal pathogen, Ascosphaera apis. Journal of Apicultural Research: Vol. 54, No. 2, pp. 105-107.

Chronic infections can profoundly affect the physiology, behavior, fitness and longevity of individuals, and may alter the organization and demography of social groups. Nosema apis and Nosema ceranae are two microsporidian parasites which chronically infect the digestive tract of honey bees (Apis mellifera). These parasites, in addition to other stressors, have been linked to increased mortality of individual workers and colony losses in this key pollinator species. Physiologically, Nosema infection damages midgut tissue, is energetically expensive and alters expression of immune genes in worker honey bees. Infection also accelerates worker transition from nursing to foraging behavior (termed behavioral maturation). Here, using microarrays, we characterized global gene expression patterns in adult worker honey bee midgut and fat body tissue in response to Nosema infection. Our results indicate that N. apis infection in young workers (1 and 2 days old) disrupts midgut development. At 2 and 7 days post-infection in the fat body tissue, N. apis drives metabolic changes consistent with energetic costs of infection. A final experiment characterizing gene expression in the fat bodies of 14 day old workers parasitized with N. apis and N. ceranae demonstrated that Nosema co-infection specifically alters conserved nutritional, metabolic and hormonal pathways, including the insulin signaling pathway, which is also linked to behavioral maturation in workers. Interestingly, in all experiments, Nosema infection did not appear to significantly regulate overall expression of canonical immune response genes, but infection did alter expression of acute immune response genes identified in a previous study. Comparative analyses suggest that changes in nutritional/metabolic processes precede changes in behavioral maturation and immune processes. These genome-wide studies of expression patterns can help us disentangle the direct and indirect effects of chronic infection, and understand the molecular pathways that regulate disease symptoms.

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.

We developed a new method for detection of the intracellular parasite, Nosema ceranae, one of the most economically devastating pathogens of the honeybee. The SWP-32 antibody was used for the development of an enzyme-linked immunosorbent assay (ELISA). We also compared the efficiency of this ELISA to microscopy and quantitative real-time (qRT) PCR, the methods currently in use. ELISA is comparable in sensitivity with the qRT-PCR, less expensive and faster. When this method is commercialized and made available to bee-keepers, it will allow them to make informed decisions for the application of in-hive chemicals. Hence, bee-keepers may be able to determine when treatments for control of N. ceranae are unnecessary and reduce the cost, time and possible side effects of these treatments. This assay provides the first serological method for detection of N. ceranae in bee colonies, which is as sensitive as DNA amplification. It can be easily adopted for both laboratory and field applications.

Background: We present a comprehensive transcriptome analysis of the fungus Ascosphaera apis, an economically important pathogen of the Western honey bee (Apis mellifera) that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture. Results: We compared A. apis transcriptome sequence from mycelia grown on liquid or solid media with that dissected from host-infected tissue. 454 pyrosequencing provided 252 Mb of filtered sequence reads from both culture types that were assembled into 10,087 contigs. Transcript contigs, protein sequences from multiple fungal species, and ab initio gene predictions were included as evidence sources in the Maker gene prediction pipeline, resulting in 6,992 consensus gene models. A phylogeny based on 12 of these protein-coding loci further supported the taxonomic placement of Ascosphaera as sister to the core Onygenales. Several common protein domains were less abundant in A. apis compared with related ascomycete genomes, particularly cytochrome p450 and protein kinase domains. A novel gene family was identified that has expanded in some ascomycete lineages, but not others. We manually annotated genes with homologs in other fungal genomes that have known relevance to fungal virulence and life history. Functional categories of interest included genes involved in mating-type specification, intracellular signal transduction, and stress response. Computational and manual annotations have been made publicly available on the Bee Pests and Pathogens website. Conclusions: This comprehensive transcriptome analysis substantially enhances our understanding of the A. apis genome and its expression during infection of honey bee larvae. It also provides resources for future molecular studies of chalkbrood disease and ultimately improved disease management.

We investigated the effect of the parasitic mite Varroa destructor on the immunological and nutritional condition of honey bees, Apis mellifera, from the perspective of the individual bee and the colony. Pupae, newly-emerged adults and foraging adults were sampled from honey bee colonies at one site in S. Texas, USA. Varroa‑infested bees displayed elevated titer of Deformed Wing Virus (DWV), suggestive of depressed capacity to limit viral replication. Expression of genes coding three anti-microbial peptides (defensin1, abaecin, hymenoptaecin) was either not significantly different between Varroa-infested and uninfested bees or was significantly elevated in Varroa-infested bees, varying with sampling date and bee developmental age. The effect of Varroa on nutritional indices of the bees was complex, with protein, triglyceride, glycogen and sugar levels strongly influenced by life-stage of the bee and individual colony. Protein content was depressed and free amino acid content elevated in Varroa-infested pupae, suggesting that protein synthesis, and consequently growth, may be limited in these insects. No simple relationship between the values of nutritional and immune-related indices was observed, and colony-scale effects were indicated by the reduced weight of pupae in colonies with high Varroa abundance, irrespective of whether the individual pupa bore Varroa.

Diseases and other stress factors working synergistically weaken honey bee health and may play a major role in the losses of bee populations in recent years. Among a large number of bee diseases, chalkbrood has been on the rise. We present here the experimental identification of honey bee genes that are differentially expressed in response to infection of honey bee larvae with the chalkbrood fungus, Ascosphaera apis.