Location:2009 Annual Report
1a. Objectives (from AD-416)
1) Develop improved understanding of factors weakening hive vigor and foraging efficiency, and provide economically sound integrated pest management (IPM) approaches to lessen the effects of these factors. Improve IPM tactics for control of key pests of honey bees, including Varroa mites, and the small hive beetle. Develop IPM strategies to lessen pesticide/antibiotic use in managed honey bee colonies, biorational compounds, and sustainable agricultural practices/IPM tactics for use in crop production that will lessen bee exposure to pesticides. 1A) Develop IPM tools and methodologies for control of key pests, and miticide resistance management programs to preserve useful chemical options. 1B) Determine the impact of the small hive beetle on colony development and longevity, and develop management systems for controlling the beetle in hives, including use of antifeedants for protection of protein supplements from small hive beetle damage. Develop effective control programs for management of small hive beetle in bee hives, with the goal to prevent contamination of bee products. 1C) Determine impacts of pesticides on foragers, both acute lethal effects and sub-lethal effects on bee behavior due to chronic exposure, and develop methods to mitigate bee losses due to pesticides, including management strategies for minimizing exposure of bees to pesticides in the field. 2) Use molecular approaches to investigate the physiological basis for bee immune responses to fungal pathogens such as chalkbrood, and develop strategies for controlling natural honey bee diseases. Identify molecular bases for honey bee physiological responses to chalkbrood. Identify and assess the role of genes that could potentially be involved in the anti-fungal activity.
1b. Approach (from AD-416)
Objective will be achieved through development of a combination of different IPM tactics (e.g., soft pesticides, acaricide rotation program, traps, lures) for control of pests, parasites, and diseases of the honey bee, and protection of hive products. It will also involve molecular studies to better understand the genetic basis of insect resistance to the fungal pathogen Ascosphaera apis, the causative agent of chalkbrood disease in honey bees. We will conduct a genome–wide screening of the honey bee immune cDNAs and will monitor the expression profile of larval genes by direct comparison of immune vs. pre-immune cDNAs. We will then utilize qRT-PCR approach to monitor expression profiles of the selected genes, identified through the genomic screens of bee's cDNAs, to better understand the correlation between changes in the level of gene transcripts and the progression of the disease.
3. Progress Report
Among a large number of bee diseases, chalkbrood has been increasingly on the rise. This disease is caused by the pathogenic fungus Ascosphaera apis. Although this disease does not normally kill an entire bee colony, it may cause rapid mortality of the brood, leading to deterioration of an infected colony. We use molecular approaches to investigate the physiological basis for bee immune responses to fungal pathogens such as chalkbrood, and we also develop strategies for controlling natural honey bee diseases. In this study, we analyzed the honey bee transcriptional response to A. apis, an infection, using in vitro larval bioassay and natural mode of larval infection. Differentially expressed transcripts were identified in infected vs. healthy honey bee larvae. These transcripts were cloned, sequenced, and analyzed. Based on the sequence analysis, a set of PCR primers were designed to amplify the most promising molecular immune-related targets. These primers are now being tested with the intent to use them in the functional studies to examine honey bee immune defenses against fungal pathogens. We have also analyzed the genome of the honey bee bacterial pathogen, Paenibacillus larvae, the causative agent of American Foulbrood disease in bees and identified a large number of genes potentially involved in the disease pathogenesis. We are now in the process of constructing bacterial mutant strains containing or lacking virulence factors. This will allow us to conduct studies to test which of the virulence genes are functionally important in infection of the host. A large-scale field trial was conducted near Wasco, CA, to determine the rate of Nosema ceranae infection in bee colonies. Some individual bees had >200 million spores, which is probably a new record. The effect of several experimental honey bee diets on the nutritional health of honey bees was tested near Modesto, CA, and compared with three currently available diets. One of the experimental diets appears promising. At the time of testing, there were large cost differences of available diets. A preliminary cost/benefit examination did not reveal a significant benefit for the most expensive diet.
1. Honey bee transcriptional response to chalkbrood disease: Chalkbrood, a major honey bee disease, is widespread, and understanding honey bee immune responses to this disease has become increasingly important. Researchers at the Honey Bee Research Unit, Weslaco, Texas, have completed analyses of honey bee genomic response to fungal infection in bees using in vitro larval bioassay and natural mode of infection. Differentially expressed genes were identified in infected vs. healthy honey bee larvae. Fragments of these genes were cloned, sequenced, and analyzed. Based on the sequence analysis, a set of PCR primers were designed to amplify the most promising molecular immune-related targets. These primers are now being tested with the intent to use them in the functional studies to examine honey bee immune defenses against fungal pathogens. This research will lead to a better understanding of honey bee responses to pathogenic fungus, which will lead to improved disease management techniques and improved strength of bee colonies.
2. Efficacy of operational device containing brood pheromone in honey bee colonies: Scientists at the Honey Bee Research Unit conducted a small-scale field experiment in Weslaco, Texas, to test efficacy of the brood pheromone devices (small plastic pouches containing brood pheromone) in overwintering honey bee colonies. Assessment of colony strength was conducted at the start and end of the study. All colonies were measured for amount of adult bees and brood area. Ratios of pollen to non-pollen forages and pollen load weight were monitored and recorded weekly. Substantial increase of adult and brood population was recorded in all treatments at the end of the study. Growth of the overwintering colonies was very robust. All treatments showed statistically significant improvement in colony strength at the end of the study. Treated colonies performed better in terms of increased number of brood, amount of pollen collected by forages, and increase in consumption of the supplemental diet. Controlled release of the stabilized brood pheromone can potentially enhance colony growth and vigor, improve pollination, and therefore increase crop yield.
3. Successful introduction of DNA into American Foulbrood natural plasmids: Scientists at the Honey Bee Research Unit, Weslaco, Texas, recently identified several new plasmids in American Foulbrood (AFB) bacteria and created a set of several unique insertions (known DNA sequences) into these natural Paenibacillus larvae plasmids. Plasmids are typically circular dsDNA molecules that very often carry antibiotic resistance and/or toxin genes, and are therefore very important in terms of host invasion and resistance to antibiotic drugs. The insertions will allow DNA sequencing of the entire plasmids, which may yield information relevant to the ability of bacterial strains caring these plasmids to infect honey bee larvae. This study is a step toward developing a genetic system for AFB bacteria.
4. A simulation of conditions associated with a bee kill during fenpyroximate application in California: Almond growers apply fungicides when rainy, moist conditions are expected, in order to prevent blossom rot. One of the more commonly used fungicides for this is Vanguard. Scientists at the Honey Bee Research Unit in Weslaco, Texas, discovered that Vanguard interacts with the active ingredient in Hivastan (fenpyroximate), a newly registered product for the control of Varroa destructor, the most important honey bee parasite in the US. We identified this interaction as a probable cause for the large bee kill. Almond growers need to exercise care in applying this fungicide during the pollination period if colonies are being treated for varroa with Hivastan. Alternatively, colonies could be treated prior to bloom. Colony stressors like the parasitic mite varroa, medications to control it, and pesticide exposure can seriously compromise honey bee health, especially if substandard nutrition has already weakened honey bees. Rapid dwindling frequently occurs and, if severe, can result in complete colony collapse.
5. Interaction of varroa parasite levels and acaricide treatments resulting in adult bee death: When some acaricides are applied to colonies for varroa control, colonies show high levels of variability with respect to adult bee mortality during the first 72 hours of treatment (e.g., 10 – 6,000). Our working hypothesis is that the number, kind, and magnitude of stressors acting on a colony prior to the application of an acaricide are the reason. We have found that pretreatment varroa levels are highly correlated with the number of adult bees that die. Colonies with high levels of mites should probably be treated with a mild acaricide first. Only after colonies are on the way to recovery and mite levels, while still serious, are not colony threatening, should stronger acaricides be used. It's likely that this principle will hold for other stressors, e.g., a pesticide spray on a pollinated crop is likely to kill more bees in colonies whose health is compromised in some way.
Murray, K.D., Aronstein, K.A. 2008. Transformations of the Gram-positive honey bee pathogen, Paenibacillus larvae, by electroporation. Journal of Microbiological Methods. 75(2):325-328.