1a. Objectives (from AD-416):
Chalkbrood causes significant mortality in bees, especially the solitary bees, but also in honey bees. These bees are important both economically and ecologically as pollinators of flowering plants, the source of most of our fruits and vegetables. The objective is to use chalkbrood as a model system to determine (1) the host/pathogen evolutionary relationship between bees and pathogens, and (2) determine how environmental stressors (such as pesticide exposure and inadequate diet) affect bee immunity. An improved understanding of host/pathogen evolution will help us identify potential sources of new, emergent pathogens in bees and provide better guidance to regulators regarding needed importation restrictions. Improving our understanding of the effects of stress on bee immunity will help in the development of effective disease control strategies for bees, especially bees produced for agricultural pollination.
1b. Approach (from AD-416):
A. Pathogen Evolution Work: The current phylogeny of the Ascosphaera will be expanded using sequences from 3-5 DNA-regions. The current list of species that have already been sequenced is too small to substantiate any phylogenetic connections between the host and pathogen groups. Now, we will collect chalkbrood pathogens worldwide, but in particular from Asia, and especially from Asian bees used for agricultural pollination. B. Genetics of Immunity. We previously identified a large number of immune response genes from the alfalfa leafcutting bees. Now, we will conduct high-throughput sequencing of other bees species to see if we can identify similar immune response genes in other bee groups. Gene expression techniques will be used to quantify which of these genes are being activated or inhibited by environmental stress factors, such as sublethal pesticide exposure. C. Determine effects of environment on bee health, especially honey bee health, using an epidemiological approach. Compare and evaluate existing geospatial and temporal datasets on honey bee health with those on cropping patterns, pesticide use, and other environmental factors to identify patterns associated with bee declines. The analysis will eventually be expanded to include geospatial data on native bee diversity.
3. Progress Report:
Molecular techniques were developed to improve alfalfa leafcutting bee identifications and to determine the evolutionary relationships between the Ascosphaera species. The phylogeny was evaluated using a combination of six different genetic regions of the fungal DNA, unlike previous studies that only used one. One species previously identified as Ascosphaera torchioi, was found to be distantly related to the other species, and probably does not belong to this genus. Two of the Ascosphaera fungi are commercially important because ones is a pathogen of honey bees and the other of alfalfa leafcutting bees. We found that these two are not as closely related as previously thought, and one arose from a group of pathogens, while the other arose from a group of saprophytes. Additional work was conducted to identify the genetics of bee immune responses and pesticide resistance (detoxification abilitiy). Honey bees have previously been found by others to have few genes for detoxifying pesticides and other environmental toxins (such as plant toxins), fewer genes than some other common insects like flies. We expected bumble bees to be similar to honey bees in this respect, considering that the two bees are in the same family, both are social insects, and both are pollinators of a wide variety of flowering plants. To test this idea, active genes (messenger RNA) were isolated from different life stages of the Hunt bumble bee (Bombus huntii), and we determined that this bumble bee has the genetic potential to produce a large number of enzymes that breakdown pesticides or repair damage caused by environmental stresses (including physiological injury caused by pesticides). However, the number of genes in the various biochemical pathways associated with pesticide resistance was substantially fewer than found in fruit flies, a model organism for genetic studies, and slightly fewer than are found in the honey bee. Also, many more detoxification/stress related genes were expressed in adult female bumble bees (workers and queens) than in adult males and larvae. The detoxification and stress related genes identified from Hunt’s bumble bee tended to be intermediate between those found in fruit flies and honey bees, based on gene sequence similarity. Interestingly, the honey bee was determined to have evolved some unique, closely related genes with a wide variety of functions. Both bees developed some detoxification functions that are similar to each other, but that arose independently in each species.