Source: KENTUCKY STATE UNIVERSITY submitted to
EFFECTS OF NOSEMA CERANAE ON HONEY BEE POLLINATION AND HONEY PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
EVANS-ALLEN
Reporting Frequency
Annual
Accession No.
0231114
Grant No.
(N/A)
Project No.
KYX-10-12-47P
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Webster, T.
Recipient Organization
KENTUCKY STATE UNIVERSITY
(N/A)
FRANKFORT,KY 40601
Performing Department
Agriculture & Environmental Science
Non Technical Summary
A honey bee pathogen, Nosema ceranae, was discovered recently and is now found to be widespread in the U.S. and the world. It infects the bee's digestive system, and weakens it so that the bees are less able to perform energetic tasks. One such task is flight to flowers for the purpose of pollination. Billions of dollars worth of crops require honey bee pollination in this country annually, so the significance of this pathogen may be substantial. The proposed study would evaluate the effects of this pathogen on two crops with very different flower forms: balckberries and sunflowers. Bees will be collected from the blooming crops and at their hives for examination. Indicators of the disease and the amount of pollen they carry will indicate whether the disease is having an effect on pollination. Bees kept indoors in glass-walled "observation hives" will also be watched for the effects of the disease on characteristic behavior that pertains to pollination. The effects of the disease on the bees' visits to selected cover crops will be recorded and evaluated also. Honey production by infected and uninfected hives will be recorded and compared. Taken together, the results of this study will be communicated to beekeepers, those who grow bee-pollinated crops, and policy makers at the federal and state levels. The outcomes may include efforts to fund research on more effective agents for control of this disease. Also, movement of bees and bee hives on a commercial scale may be reconsidered in circumstances when the pathogen is moved with the hives.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21130101130100%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
3010 - Honey bees;

Field Of Science
1130 - Entomology and acarology;
Goals / Objectives
The project will evaluate the effects of a common pathogen of honey bees, Nosema ceranae, on the pollination of crops with different floral morphologies and on honey production. Fumagillin, the treatment commonly used to control the pathogen, will be evaluated for possible negative effects on the bees. This pathogen infects the midgut of the bee, destroys much of the tissue in the midgut, and interferes with the energetic requirements of the bee. Consequently, crops with small flowers requiring many short flights are likely to receive less adequate pollination by Nosema-infected bees, compared to crops with large flowers requiring less flower-to-flower flight. The objectives are to (1) evaluate the effects of N. ceranae on blackberry and sunflower pollination efficacy by honey bees, (2) evaluate the effects of N. ceranae on honey production by honey bees, (3) determine the effects of fumagillin on honey bee pollination efficacy, by infected and by healthy bees, and (4) to determine whether N. ceranae affects honey bee visits to cover and seed crops commonly grown in Kentucky. Objective 1 will be accomplished during October 2012 through September 2014. Objective 2 will be accomplished during October 2013 through September 2014. Objective 3 will be accomplished during October 2014 through September 2015. Objective 4 will be accomplished during October 2015 through September 2016. Outputs will be (a) data on the effects of the pathogen N. ceranae on the pollination of blackberry and sunflower crops, (b) data on the effects of the pathogen on honey production, (c) data on the effects of fumagillin on the pollination of these two crops, and (d) data on the effects of the pathogen on honey bee visitation to selected cover crops grown in Kentucky. Beekeepers and growers of bee-pollinated crops will have the opportunity to observe the research projects while attending Kentucky State University (KSU) Field Days, beekeeping workshops conducted by KSU apiculture research staff, and other events scheduled at the KSU research farm. KSU students will have the opportunity to participate in the projects for academic credit. The results of the study will be made available as practical knowledge for federal and state policy and decision makers.
Project Methods
Experimental hives will be established and either inoculated with the pathogen Nosema ceranae or left healthy. The hives will be near blackberry and sunflower plantings. Bees collected on blackberry and sunflower bloom, and as they return to their hives, will be examined for pollen loads and infection with the pathogen. Early and late infection levels and midgut strutural damage will be determined by light microscopy. Correlations between infection and pollen load weight will be determined. Bees will be inoculated and marked, and bees not inoculated will be marked with a different color. These bees will be collected at later dates when leaving their hives, and examined for infection to determine the effect of infection on flight behavior. Similarly, bees that have been inoculated and not inoculated will be introduced to observation hives and later observed for their participation in waggle dance activities. Outdoor hives will be inoculated or not inoculated, and compared for their honey production. The effects of fumagillin, the approved treatment for this pathogen in honey bees, will be evaluated in terms of its effects on pollination behavior, compared to pollination behavior of bees not fed fumagillin. The effects of the pathogen on nectar and pollen collection on selected cover crops will be evaluated, according to the format described above.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The target audiences are Kentucky beekeepers and those in Kentucky who grow honey bee-pollinated fruits and vegetables. Changes/Problems:The PI experienced serious health problems in 2016 and 2017, and was unable to retain experienced beekeeping help. Consequently, the goals were changed to laboratory-based goals. What opportunities for training and professional development has the project provided?This work allowed me to attend and learn from the 2017 American Bee Research Conference and the Society for Invertebrate Pathology Annual Meeting in 2017. Both allowed me to converse with colleagues and learn much about their discoveries and methods. How have the results been disseminated to communities of interest?These results have been communicated to beekeepers who held the American Beekeeping Federation conference coincident with the American Bee Research Conference. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The PI experienced serious health problems in 2016 (heart attack) and 2017 (kidney surgery). In addition, we were unable to retain beekeeping help preventing the PI from reaching the above goals, which would have required extensive beekeeping and other outdoor work. No other permanent staff had the ability to work with bees. Instead, we continued laboratory studies on Nosema ceranae infection in caged bees, These included: (a) the improvement of methods for embedding, slicing and staining healthy and Nosema-infected midguts, (b) staining methods for Nosema spore and everted polar filaments, and (c) effects of gluconic acid as a treatment for Nosema. For (a) we found that 10% sodium dodecyl sulfate clarified worker bee midguts much more effectively and reliably than the KOH solutions used previously. This method now allows clear imaging of the peritrophic matrix as well as particles (such as spores and pollen grains) in the midgut lumen. We also evaluated a large number of stain combinations for histological sections of the honey bee midgut. Alizarin red and calcofluor white make the most effective combination. Alizarin red highlights the midgut tissue and the peritrophic matrix, while calcofluor white stains the spores in the midgut cells. Intensity of calcofluor staining indicates the maturity of the spores, because the thickness of the spore's chitin exterior determines the amount of calcofluor that binds to the spore. After inoculating individual bees with known spore dosages of 20,000 spores in sucrose syrup, we were able to distinguish the progress of the infection with these tools. At 9 days post-inoculation, discrete clusters of spores were clearly visible within midgut cells. Since these clusters are not contiguous, it seems that each cluster represents a single infection site. Hence, we should be able to estimate the number of successful infective spores for a single worker bee. We expected to see infections mainly in the midgut cells closest to the midgut lumen because these cells should be more easily reached by the everted polar filaments. However, we were surprised to see that the entire length of the midgut epithelial cell layer contained a nearly equal distribution of spore clusters. Even the tight invaginations of the midgut included many spores. The reason for this nearly equal distribution is not clear. At 10 days post-inoculation, the spore clusters were more diffuse, as cell-to-cell infections increased the number of infected cells. At 14 days post-inoculation, the entire midgut was infected, including cells adjacent to the basement membrane. For (b) we have had success staining spores with DRAQ5. This is a cell-permeant stain that has been used to follow initial infections by microsporidia closely related to Nosema. Also, we found that coomassie orange binds to the glycoproteins in the everted polar filament. We hope that this will lead to a method for quantifying the degree of spore germination as stimulated on microscope slides. For (c) we have found encouraging early results that suggest a role for gluconic acid in inhibiting Nosema in caged bees. These studies will be replicated in the coming years.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: W. Li, J. D. Evans, Q. Huang, C. Rodr�guez-Garc�a, J. Liu, M. Hamilton, C. M. Grozinger, T. C. Webster, S. Su, Y. P. Chen. 2017. RNA interference (RNAi) as a novel treatment for Nosema ceranae infection in European honey bees Apis mellifera in Proceedings of the American Bee Research Conference. Bee World, 93(4): 113.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: T. C. Webster, M. Matisoff, K. Kamminga, C. Butler. 2017. Secretion of the peritrophic matrix in the honey bee, Apis mellifera, midgut is impaired by Nosema ceranae infection. Presented at Society for Invertebrate Pathology 2017, La Jolla, CA, August 13-17, 2017.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audiences are Kentucky beekeepers and those in Kentucky who grow honey bee-pollinated fruits and vegetables. Changes/Problems:A graduate student responsible for maintaining bee hives and collecting data resigned, so that the project had no beekeeping assistance. Many of the university bee colonies died because of this resignation and because the colonies apparently had diseased or infertile queen bees. The program recently hired a research assistant who will learn beekeeping and assist the project. In addition, we will attempt to purchase better-quality queen bees for the university colonies so that the hives can be used for the planned project. What opportunities for training and professional development has the project provided?This project allowed me to attend the American Bee Research Conference, where North American honey bee scientists present their work and develop collaborations. How have the results been disseminated to communities of interest?The results were presented at the American Bee Research Conference in January 2016 and in peer-reviewed scientific publications. What do you plan to do during the next reporting period to accomplish the goals?We recently hired a new research assistant who will learn beekeeping and assist the PI in completing the project.

Impacts
What was accomplished under these goals? Improved methods for the examination of honey bee tissues were developed. These include clarification of the bee's midgut tissues with potassium hydroxide (KOH) solution so that a critical structure, the peritrophic matrix, can be examined by fluorescence microscopy. Degradation of the peritrophic matrix may be caused by Nosema ceranae infection, with consequent effects on the bee's nutritional status and ability to pollinate crops. A method for creating 3-dimensional images of the midgut, and including the peritrophic matrix, was developed. This allows us better opportunities to assess the effects of Nosema ceranae infection.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Cameron J. J., H. M. Lucas, T. C. Webster, R. R. Sagili. 2016. Colony level prevalence and intensity of Nosema ceranae in honey bees (Apis mellifera L.) PLOS ONE http://dx.doi.org/10.1371/journal.pone.0163522
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Webster, T. C., M. A. Matisoff, and C. Butler. 2016. Nosema spore detection by optical methods American Bee Research Conference, January 8-9, 2016, Jacksonville, FL.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Webster, T. C. in press. Dysentary. In ABC and XYZ of Bee Culture. A. I. Root, Medina OH.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Matisoff, M. A., T. C. Webster and C. Butler. 2016. Nosema polar tube morphology suggests infective mechanisms within honey bee midguts (Apis mellifera). American Bee Research Conference, January 8-9, 2016, Jacksonville, FL.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Butler, C., M. A. Matisoff, and T. C. Webster. 2016. New histological methods improve diagnostic techniques for identifying Nosema in the honey bee midguts. American Bee Research Conference, January 8-9, 2016, Jacksonville, FL.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The target audience is Kentucky beekeepers and those who grow bee-pollinated crops. In addition, KSU students have learned laboratory techniques pertaining to microscopy, polymerase chain reaction (PCR), microbiology, and pathology. Changes/Problems:I am obliged to perform all of the beekeeping activities that pertain to the objectives listed in the proposal. This includes establishing the hives, adding hive boxes when needed, monitoring mite infestations, treating hives as needed for mite infestations, feeding hives when they are established and must be sustained during dearth periods, re-queening hives as needed, introducing inoculated bees to hives, weed-eating around the hives to suppress weeds, and preparing hives for winter. What opportunities for training and professional development has the project provided?The project has allowed the PD (Webster) to attend and present research at the January 2015 American Bee Research Conference. At this conference we all discussed our research, possible collaborations, and how this all relates to advancement in American beekeeping. How have the results been disseminated to communities of interest?The results were disseminated at the 2015 American Bee Research Conference, the Beekeepers of Tennessee conference (Cookeville TN, October 2014), the Young Harris Beekeeping Institute (Young Harris GA, May 2015), and the Heartland Apicultural Society (Albion MI, July 2015). What do you plan to do during the next reporting period to accomplish the goals?We expect to use our laboratory and software techniques to implement the goals 1 through 4 above.

Impacts
What was accomplished under these goals? A transect along the margin of the KSU research farm was planted in buckwheat, a crop attractive to honey bees. Four hives were established at the northern end of the transect. The hives were established so that worker honey bees could be inoculated with Nosema ceranae spores and then introduced to the hives. Nosema ceranae was identified by students in infected honey bees with primers specific to the pathogen using the Polymerase Chain Reaction. This work pertains to objective 4 above. In addition, we have adapted a software program so that it will count spores extracted from bees. The software recognizes the specific shape and color of each spore in a micrograph, and counts them. This method will allow us a more rapid and objective method of assessing Nosema ceranae infections in our experimentally inoculated bees. It will help other researchers at other institutions who also study this pathogen and who must quantify infections.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Rogers, Kemper, Thomas Webster, and Li Lu. (2014) Identification of Nosema pathogens in honey bee with Polymerase Chain Reaction. Kentucky Academy of Science Meeting, Lexington Kentucky, November 15, 2014.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Davie, Alexander, Thomas Webster, and Li Lu. (2014). Analyze the Honey Bee Epidemic with Molecular Methods. Kentucky Academy of Science Meeting, Lexington Kentucky, November 15, 2014.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Conrad, Kristin, Thomas Webster, and Li Lu. (2014). A study of Nosema apis and Nosema ceranae in Honeybees, Apis mellifera. Kentucky Academy of Science Meeting, Lexington Kentucky, November 15, 2014.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audiences are researchers and extension professionals who investigate honey bee diseases and related maladies of honey bees; and beekeepers who may keep bees infected with honey bee pathogens. Efforts were formal communications at beekeeping workshops and conferences, and informal communications with beekeepers. Changes/Problems: We have had some difficulties in propagating the Nosema cerane pathogen, using caged bees. We are perfecting our methods of storing the N ceranae spores and inoculating bees, so that sufficient inoculum will be available for the project in this coming season. What opportunities for training and professional development has the project provided? The project has allowed a Master's of Science in Environmental Studies graduate student to learn and perfect the realtime PCR technique. This includes the method for extracting and amplifying Nosema DNA from infected bees. How have the results been disseminated to communities of interest? Hives belonging to commercial beekeepers have been monitored for Nosema disease and we have discussed the results with them. The hive infection rate in 2014 was significantly lower than the rate in 2013. We have discussed the possible reasons for this decline in infection, but have not reached any conclusions. Updates on this project have been presented at a scientific conference to honeybee research scientists. Updates on this project were also discussed at several bee keeping workshops in Kentucky to stakeholder producers. What do you plan to do during the next reporting period to accomplish the goals? We plan to complete most or all of the objectives 1, 2, 3 and 4.

Impacts
What was accomplished under these goals? The realtime polymerase chain reaction is a molecular biology technique based on the polymerase chain reaction (PCR). PCR can be used to amplify and detect or quantify a specific DNA molecule of interest, such as DNA of a pathogen in a host organism. For Objectives 1, 2, and 4: The method for realtime PCR analysis of Nosema-infected bees was developed and perfected using a realtime PCR thermocyler. A Master's of Environmental Studies graduate student optimized this realtime PCR method and is currently examining Nosema infection biology in honeybees as part of his thesis research. After optimization, this technique was able to detect very low infection levels in honeybees in a quantitative manner, prior to any visual change in the honeybee digestive system. This technique is now being implemented in a series of experiments. For Objective 1: A transect measuring 6 m by 800 m was prepared at the KSU Research Farm and planted with buckwheat, a nectar-producing crop attractive to honey bees. This crop will be replanted on this site in 2015. Observations of foraging bees on this transect will supplement observations of bees on blackberry and raspberry crops at the KSU farm.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Webster, T., M. Matisoff & C. Butler 2014. Midgut morphological changes that accompany Nosema ceranae infection in honey bees. American Bee Research Conference, January 11, 2014, San Antonio, TX. Abstract published in American Bee Journal, 154(5).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Matisoff, M., C. Butler & T. Webster 2014. Honey bee midgut images using histological techniques. American Bee Research Conference, January 11, 2014, San Antonio, TX. Abstract published in American Bee Journal, 154(5).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Vance, D., L. Lu & T. Webster. Identification of Nosema ceranae Pathogen in Bee Hives of Kentucky Area with Molecular Methods. Annual Conference of the Kentucky Academy of Science, Morehead, KY, November, 2013
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lu, L. & T. Webster. Molecular Differentiation of Nosema apis and Nosema ceranae Based on Species-Specific Sequence Differences in 16S rRNA and RPB1 Genes. Annual Conference of the Kentucky Academy of Science, Morehead, KY, November 2013.


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audience is primarily (1) scientists who study honey bees, honey bee diseases, horticulture, and related subjects, (2) apiary inspectors who advise beekeepers on control of the pathogen Nosema ceranae, and (3) beekeepers who provide hives for crop pollination. To a lesser extent state and federal agencies and the general public will be advised of the results because all Americans benefit from consuming bee-pollinated crops. Changes/Problems: No changes or problems have occured for this project. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Results to date have been presented to the annual meeting of the American Association of Professional Apiculturists, which met in Hershey PA in January 2013. This conference was held in conjunction with annual meetings of the Apiary Inspectors of America and the American Beekeeping Federation. Consequently, all three groups were able to attend my presentations. These three groups comprise many of the honey researchers, state hive inspectors, and professional beekeepers, respectively. What do you plan to do during the next reporting period to accomplish the goals? We plan to (1) establish plots of sunflowers and work with colleagues at KSU to maintain existing blackberry bushes, (2) to establish an apiary near these plots, (3) to infect the bees in these hives with Nosema ceranae, with an expectation that not all of the bees will be equally infected, (4) to evaluate the effects of plant pollination behavior relative to N. ceranae infection levels, and (5) to calculate statistical correlations between these variables

Impacts
What was accomplished under these goals? Under these goals we have developed new methods of staining and histology which shed light on the development of N. ceranae in honey bees. These methods will help us to evaluate the effects of this pathogen on honey bee pollination efficacy. In particular, we have found (1) that the peritrophic matrix (a structure lining the midgut of most insects) encloses many of the N. ceranae spores in the midgut although some spores must have bypassed this matrix to infect midgut epithelial cells; (2) that the N. ceranae infection can proceed to the outermost cells of the midgut, adjacent to the hemolymph (lending credence to the idea that the hemolymph transports this pathogen to other tissues in the bee); and (3) that peritrophic matrix secretion can be observed clearly using fluorescence microscopy at 1000x with appropriate staining techniques, and that this secretion is clearly localized to certain midgut epithelial cells.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Webster, Thomas. C. and Martin A. Matisoff. 2013. Early development of Nosema ceranae in honey bee midgut tissue. American Bee Journal 153(4)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Matisoff, Martin A. and Thomas C. Webster. 2013. An inexpensive test for Nosema in honey bees. American Bee Journal 153(4)