Source: PENNSYLVANIA STATE UNIVERSITY submitted to
IMPACTS ON HONEY BEES AND HONEY BEE DISEASES FROM IN-HIVE MITICIDE USE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0213374
Grant No.
2008-37610-18842
Project No.
PEN04235
Proposal No.
2008-00512
Multistate No.
(N/A)
Program Code
NI
Project Start Date
Sep 1, 2008
Project End Date
Aug 31, 2010
Grant Year
2008
Project Director
Ostiguy, N.
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802
Performing Department
ENTOMOLOGY
Non Technical Summary
This proposal addresses honey bees health. The accessibility of managed bees and the background pollination by feral colonies has declined over the past ten years due largely to varroa mites and viruses. It is extremely important that the causes of Colony Collapse Disorder (CCD) be found. A correlation between the occurrence of a newly identified virus (Israeli Acute Paralysis) and CCD has been observed but a number of other disease organisms are also found in the dying colonies. We suspect more than one factor is causing colony collapse. Other factors could include in-hive miticides, pesticides collected by bees while foraging, poor nutrition, and stress. The three objectives addressed are to evaluate the impact of in-hive miticides application on 1) viruses of honey bees including prevalence, and titer, 2) honey bee immune function, and 3) longevity, health, and reproductive physiology. Colonies will be randomly assigned to four treatments u control, CheckMite<, Apistan< and formic acid-flash treatment. When one colony reaches the mite threshold, treatments will be applied. Bees will be taken for viral and immune response analysis when packages are installed, immediately prior to miticide treatment and for six week following the initiation of treatment. Viral identification and viral, antimicrobial peptides and immunity-related enzymes quantification will be completed following standard laboratory protocols. Longevity of exposed and unexposed eggs, larvae, pupae and adults will be studied by sampling and recording the number of each at appropriate intervals. Emerging adults and mites will be collected and housed in separate cages determined by mite presence/absence. At death or at the end 1 week, bees will be analyzed for viruses, anti-microbial peptides, and immune-related enzymes using PCR. Sperm viability will be assessed in acute and chronically exposed and unexposed drones. Miticide effects will be assessed on sperm viability in the spermathecae of queens instrumentally inseminated with sperm from treated drones. Quality and survival of brood plus effects of miticides on queen production, survival and supersedure will be evaluated. No decline in colony prevalence or mean titers is anticipated following miticide applications unless viral prevalence exceeds 50%. A relationship is expected between suppression of immune-related genes and varroa infestation and in miticide treated colonies. After exposure is removed, mean suppression of immune-related genes in a colony is expected to diminish. The greatest longevity is anticipated in bees tested in late spring because these bees have the least varroa, lowest viral titers and least immunosuppression. The shortest longevity is anticipated in bees after miticides have been applied u higher mite levels, greater immunosuppression, higher stress. A greater impact on sperm viability and sperm viability in the spermathecae is expected when exposure is to fluvalinate or coumaphos. The results of this work should give beekeepers clear information about the possible adverse impacts from the application of various miticides.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013010113015%
3063010109015%
3063010110115%
3113010113015%
3113010115010%
3113010117015%
3143010113015%
Goals / Objectives
The three objectives addressed by the projects are: 1) Evaluate the impact of miticides on viruses of honey bees, 2) Evaluate the impact of miticides on honey bee immune function and 3) Evaluate the impact of miticides on honey bee longevity, health, and reproductive physiology. Several honey bee viruses are vectored by the parasitic mite, Varroa destructor. One might infer that the colony prevalence of varroa vectored honey bee viruses would decline subsequent to the application of a miticide. It has been observed that DWV prevalence and titer may increase following a late summer or early fall miticide treatment. These treated colonies have been observed to collapse even when mite numbers have been reduced significantly below the economic threshold. To prevent colony loss, implementing mite control earlier in the season, before viral prevalence or titers are elevated, may be more effective and necessary. The direct impact of miticides on viral prevalence, titer or spread has not been described. . Stress, a factor known to influence immune function, can result from chemical exposure. Our preliminary data show an increased prevalence of a virus after colony exposure to a miticide. We postulate this may be due to the miticide compromising honey bee immune function. Many factors influence longevity and health of workers and drones and bee reproductive physiology. Reduced longevity and sub-optimal learning are potential outcomes of miticide exposure. Imidacloprid has been reported to adversely impact honey bee learning and other pesticides may have similar impacts. Preliminary data show a decrease in sperm viability when drones are exposed to miticides such as coumaphos. Increased sperm mortality over time may be a factor leading to queen failure. No decline in colony prevalence or mean titers is anticipated following miticide applications unless viral prevalence exceeds 50%. A relationship is expected between suppression of immune-related genes and varroa infestation and in miticide treated colonies. After exposure is removed, mean suppression of immune-related genes in a colony is expected to diminish. The greatest longevity is anticipated in bees tested in late spring because these bees have the least varroa, lowest viral titers and least immunosuppression. The shortest longevity is anticipated in bees after miticides have been applied; higher mite levels, greater immunosuppression, higher stress. A greater impact on sperm viability and sperm viability in the spermathecae is expected when exposure is to fluvalinate or coumaphos.
Project Methods
Twenty-eight new colonies will be randomly assigned to a treatments - control, CheckMite, Apistan and formic acid-flash treatment. When one colony reaches 10 mites/day on a sticky board in June/July or 50 mites/day in Aug/Sept, miticide treatments will be implemented. Controls will be given a sham treatment. Colony health will be assessed (size, chalkbrood, American Foulbrood (AFB), queen cells and replacement and colony survivorship). Beginning one week after bees are installed, 3-day sticky boards be used continuously to monitor for varroa. Bee samples from each colony will be taken for viral and immune response analysis when packages are installed, immediately prior to miticide treatment and for six week following treatment initiation. Samples are placed in TRIazol and stored at -80C. Viral identification and viral, antimicobial peptide and immunity-related enzymes quantification will be completed following standard laboratory protocols, including a negative, a positive and an internal control. Longevity of exposed and unexposed eggs, larvae, pupae and adults will be determined by confining queens on drawn comb (worker and drone). Eggs, larvae and pupae will be sampled at appropriate intervals and the number of each will be recorded. One frame of emerging adult bees will be removed from a colony in each treatment. Emerging adults and mites will be collected. Four replicates (2 parasitized and 2 non-parasitized) of 15 newly emerged adults will be placed in cages. Dead bees will be counted every 6 hours for 1 week and analyzed for viruses, anti-microbial peptides, and immune-related enzymes using PCR. Miticides effects on sperm viability will be screened. The effects of miticides on the viability of sperm found in the spermathecae of queens instrumentally inseminated with sperm from treated drones will be evaluated. Brood quality and survival will be monitored along with queen production, survival and supersedure. Colony prevalence and/or mean virus titers, and immune response measures will be used as outcome variables in a multi-factor ANOVA or MANOVA while miticide treatment and mite level at time of miticide application will be used as predictor variables. Mite levels, virus prevalence and mean titers, and suppression of immune-related genes and miticide treatment are all potential predictor variables of honey bee longevity and survivorship of eggs, larvae and pupae. Survival analysis is expected to produce the clearest understanding of the data. Sperm viability (drones and from spermathecae) will be assessed using a two-way repeated measures ANOVA and/or survival analysis with miticide treatments and time as independent variables. MAAREC, a consortium of beekeepers from state associations, regulators and researchers, meets twice each year to discuss current and emerging issues of concern to beekeepers and to disseminate results. The study results will give beekeepers clear information about the possible adverse impacts from the application of various miticides.

Progress 09/01/08 to 08/31/10

Outputs
OUTPUTS: Several outputs relate to this project. A queen cage, allowing for accurate determination of the age of eggs on a full frame of comb, has been developed and tested. This device confines a queen on a frame while she lay eggs on both sides and allows the free movement of workers in and out of the cage. Once the queen has been removed from the cage all eggs will be the same age, which is critical for determining the % of eggs hatched. A second method developed is the accurate assessment of the egg-larvae-pupae-adult survivorship. A photographic method for recording presence/absence of each life-stage has been developed. Each successive image is overlaid on the previous image to determine survivorship of each life-stage. Only 65% of colonies in our experiments survived one summer and none survived winter. Queens were removed from failing colonies. The heads, thorax and abdomens of all queens were positive for Deformed wing virus (DWV) and negative for Black queen cell virus (BQCV) and 92% negative for Sacbrood virus (SBV). Most Eggs were positive (67%) for DWV but negative for BQCV and SBV. No Israeli acute paralysis virus was detected in any sample. Even before the experiment began many workers were positive for virus - 50% for BQCV, over 90% for DWV and over 15% for SBV. By August 50% of the bees were positive for SBV, 65% for DWV and 33% for BQCV. These were the surviving adult bees. The presence of virus in all colonies, including those established from packages in 2010 presented difficulties. [Virus results for 2010 are preliminary but look, at this time, as if they do not differ from 2009.] We have preliminary information indicating a reduction in the survivorship of eggs in colonies infected with virus. In Italian bees, the strain used in our experiments, egg survivorship is expected to be approximately 94%. We are observing a lower survivorship in the range of 75-85%. Coumaphos, survivorship seems to be lower - 65-75%. Some decline in survivorship prior to the application of miticides may be due to pesticide residues in foundation wax or pesticides collected by foraging bees. Samples of food and wax have been collected and sent for analysis. We have not seen any differences from the expected survivorship of larvae (86%) or pupae (85%). One graduate student and 6 undergraduates students were mentored; three have graduated and two will graduate this year. One graduating student, Josh Hibit, is the Dutch Gold Honey Undergraduate Scholarship recipient. My lab continued its outreach efforts with the Great Insect Fair in 2010. The PA Honey Queen assisted in our efforts to educate the public on honey flavors. I was invited to give talks at PA Certified Organic Annual Mtg 10/29/09; PA Beekeeper Assoc Annual meeting 11/19/09; Amer Bee Research Conf 1/14/10; W PA Beekeeping Seminar 2/19-20/10; Central Co Beekeepers, 5/30/10; Betterbee Field Day, 7/26/10; First Annual Pollinator Conference 7/25/10; Honey Bee Awareness Day 8/21/10; WV Beekeepers Fall Mtg 9/24-25/10; RI Beekeeping Assoc 10/17/10. PARTICIPANTS: Nancy Ostiguy (PI): responsible for the design, execution, and analysis of all experiments. Supervisor of all individuals collecting data and processing samples. Abby Kalkstein (graduate student) processed and analyzed data. Jeremy Fitzgerald (technician) managed colonies, collected and processed data. Kerry Lynott (technician & Graduate student) collected and processed data, managed laboratory. Six undergraduates (Valerie Morales, Hillary Craddock, Emily Shimp, Reagan Furbish, Josh Hibit and John Lidwell) and one graduate student (Abby Kalkstein) were provided opportunities for training and profession development. Additionally Kerry Lynott and Jeremy Fitzgerald were provided professional development opportunities. Ms Lynott and Mr. Fitzgerald participated in the 2009 and 2010 Pennsylvania State Beekeeper meeting and Ms. Lynott, Mr. Fitzerald and Mr Hibit participated in outreach activities. TARGET AUDIENCES: The target audience for this project was beekeepers, farmers utilizing honey bees for pollination, and the general public. Beekeepers, farmers and the general public include individuals from racial and ethnic minorities. Events where ethnic and racial minorities were participants include 1) farmers at the Pennsylvania Certified Organic Annual Meeting 10/29/09; 2) beekeepers and those interested in beekeeping (some of these people may not ever become beekeepers but they are interested in the topic) at the Western Pennsylvania Beekeeping Seminar 2/19-20/10; Betterbee Field Day, 7/26/10; West Virginia Beekeepers Fall Meeting 9/24-25/10; and the Rhode Island Beekeeping Association 10/17/10 and 3) the general public at The Great Insect Fair 10/2/10. PROJECT MODIFICATIONS: The use of a sheet of Plexi-glass to mark the location of eggs, followed by larvae, followed by pupae, followed by adults was eliminated from the methods as the heat generated under the Plexi-glass adversely impacted the survivorship of eggs. When this problem was addressed (by counting eggs while in the shade) the ability to accurately count the eggs became a problem and the length of time the frame of eggs was out of the hive became too long. Taking a photographic record was then substituted and after various modifications and the assistance of the Entomology Department's media specialist, Nick Sloff, we were able to make a permanent record of our observations and accurately count the egg-larvae-pupae-adult unit.

Impacts
The results from the experiments conducted from 2008-2010 have provided interesting data concerning the health of honey bee colonies. Our results are preliminary because some data analysis remains. The 2010 pesticide analysis of food and wax samples is not complete and another replicate of the coumaphos exposure experiment is planned. Our data show a small but significant difference in the egg-larvae-pupae-adult survivorship. While adverse impacts to colonies exposed to coumaphos can be demonstrated, the results are not persuasive to beekeepers yet. We wish to repeat our experiments with fluvalinate and the less toxic miticides, e.g., thymol and formic acid, to provide the beekeeper with more convincing evidence to support a change in behavior and, hopefully, evidence to support the use of less toxic miticides along with physical and cultural varroa mite control tactics. We have begun disseminating some of the information we have discovered. Foundation wax, along with comb wax, contains residues of various pesticides. The primary residues found are coumaphos and fluvalinate. We have also found higher residues of coumaphos and fluvalinate in brood comb honey that has overwintered (and is the source of carbohydrates for overwintering bees) than in the honey extracted from honey supers. Included in the information disseminated to beekeepers are the data related to the importance of a healthy queen. We have convincing evidence that queen health is related to the survivorship of eggs. We suspect that once a sufficient percentage of bees in a colony have become infected with a viruses the greater the risk of co-infection with other viruses. Until 2008-2010 DWV prevalence in colonies did not reach 60% of sampled bee until September and BQCV or SBV presence in a colony occurred in late spring and was gone by mid-summer. The data from this project indicate a change in this pattern. Bees in colonies with high prevalence of DWV are being found with both SVB and BQCV in August. We are suggesting to beekeepers that their colony management be altered. Brood comb should be no more than 3 years old. The positive impact of this management tactic is two-fold. Brood comb, which will have the highest pesticide residues in the colony, will be removed along with any food (pollen and honey) that may contain pesticide residues or be contaminated with pathogens, e.g., viruses and/or American Foulbrood. We are also suggesting that beekeepers monitor the mite levels in their colonies more closely to ensure that miticides are not being used when mite levels are low or, conversely, that mite populations are not allowed to rise above the threshold for maintaining colony health as this leads to higher levels of virus in colonies. Toward the goal of preventing the growth of mite populations we are encouraging beekeepers to breed from local queens that have survived in colonies where miticides have not been used to control mite populations, to install screened hive bottoms, to install vents or other devices to ensure proper hive ventilation, to monitor honey and pollen quantities in the hive more closely, and isolate sick colonies to reduce disease transmission.

Publications

  • Ostiguy, N. 2010. Sustainable Beekeeping: Managed Pollinator CAP Coordinated Agricultural Project - A National Research and Extension Initiative to Reverse Pollinator Decline. American Bee Journal 150(3):149-152.
  • Williams, G. R., R. E. L. Rogers, A. L. Kalkstein B. A. Taylor, D. Shutler, and N. Ostiguy. 2009. Deformed wing virus in western honey bees (Apis mellifera) from Atlantic Canada and the first description of an overtly-infected emerging queen. Journal of Invertebrate Pathology 101(1):77-79.
  • Aronstein, K., A. Averill, F. Drummond, B. Eitzer, J. D. Ellis, N. Ostiguy, S. Sheppard, M. Spivak, and K. A. Visscher. 2010. Peek at the distribution of viruses in stationary honey bee colonies in the U.S. American Bee Journal 150:132. (Abstract).


Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: A number of experiments have been completed related to the goal of determining the impact of miticide use on the survivorship of eggs, larvae, and pupae and the longevity of newly emerged adults. To ensure sufficient sample size for the longevity experiments, a frame cage was designed, built and tested. The cage, it has been determined, has no impact on the development of brood or the ability of nurse bees to complete normal brood tending tasks. We have determined the relationship between the number of minutes a frame with eggs is held outside the colony (for counting eggs or larvae) and the development rate. To not impact the development rate frames are returned to the colony within 5 minutes. A database has been developed to organize the collected data. Data may now be accessed via a secure website. It is now possible to correlate data that were housed on various individual computers. One graduate student and 5 undergraduate students have been mentored during from fall 2008 through summer 2009. Featured outputs include participation at the Great Insect Fair and Ag Awareness Day, both on the PSU University Park campus. The Great Insect Fair is an outreach event sponsored by the Entomology Department that reaches between 4,000 and 5,000 people. Visual and written materials concerning honey bee health, specific research projects (including this one), and honey varieties were developed and we presented information and conducted a honey tasting. Ag Awareness Day was an event organized by undergraduates in the College of Agricultural Sciences on April 24, 2009. My lab organized materials and people in the various the honey bee research labs at Penn State to participate in both these events. My lab also worked with the Shaver's Creek Environmental Center to develop and implement native plant and pollinator friendly demonstration gardens at Shaver's Creek. Work on this project continues with additional signage and brochures describing the various native plants and their role in the life cycle of pollinators. Preliminary work has been instigated to replicate and expand this work in the new Arboretum on the University Park campus. From October 2008 until September 2009, I was invited to give talks to four beekeeper groups and one general audience to discuss honeybees, pollination honey bee health and the research we are conducting. The talks are: Honey Bee Health: Pathogens, Disease, and Prevention, West Virginia Honey Festival, Parkersburg West Virginia August 28, 2009. Sustainable Methods of Beekeeping: Doing it without chemicals, York County Beekeepers Association, York Pennsylvania August 27, 2009. The Continuing Saga of the Honey Bee: CCD and Bee Viruses, Virginia State Beekeepers Association Leesburg Virginia April 17, 2009. Organic and Sustainable Methods of Beekeeping, Virginia State Beekeepers Association Leesburg Virginia April 18, 2009. Honey Bee Health: What is the Future of Pollination Huntingdon County Extension Annual Meeting, Huntingdon Pennsylvania February 19, 2009. Current and Future Research. Pennsylvania State Beekeeper Association, Lewisburg PA. November 7, 2008. PARTICIPANTS: Nancy Ostiguy (PI): responsible for the design, execution, and analysis of all experiments. Supervisor of all individuals collecting data and processing samples. Sheena Sidhu (graduate student): collected and processed data Jeremy Fitzgerald (technician): managed colonies, collected and processed data Kerry Lynott (technician): collected and processed data, managed laboratory Three undergraduates (Emily Shimp, Josh Hibit and John Lidwell) and one graduate student (Sheena Sidhu) were provided opportunities for training and profession development. Additionally Kerry Lynott and Jeremy Fitzgerald were provided professional development opportunities. Ms Sidhu and Ms Lynott participated in the 2008 Pennsylvania State Beekeeper meeting and Ms. Sidhu, Ms. Lynott, Mr. Fitzerald and Mr Hibit participated in outreach activities. TARGET AUDIENCES: The target audience for this project is beekeepers. Between September 2008 and August 2009, invitations from four beekeeper groups were accepted and talks followed by question and answer sessions were delivered. A secondary target audience is the general public. Three opportunities presented themselves. The West Virginia Honey Festival provided the opportunity to give a talk followed by a significant question and answer session discussing honeybees, pollination honey bee health and the research we are conducting at PSU. We also participated in several outreach opportunities in and around central Pennsylvania including the 15th Annual Great Insect Fair, Ag Awareness Day, Pennsylvania Sustainable Agriculture annual meeting, and the Shaver's Creek and PSU Arboretum native pollinator gardens. PROJECT MODIFICATIONS: There has been a delay in the collection of data specific to the application of miticides in honey bee colonies due to substantial disease problems in all the available colonies. This has resulted in potential significant data on colony health as related to the presence of pathogens. The problems experience this year should not occur as new packages will be purchased (with low mite numbers). Mite levels will not be high but this condition will be similar to that seen in beekeeping operations where the beekeeper provide prophylactic miticides treatment. This situation is relatively common among the larger beekeepers.

Impacts
The results from the experiments conducted in 2009 have provided a preliminary assessment of the health of honey bee colonies. The data are preliminary at this time because all the analyses are not completed. Currently we are waiting for the results of pesticide analyses of bee and pollen samples from the colonies in our experiment. Not all the analyses have been completed to determine the array of viruses and other pathogens in these same colonies. The preliminary results seem to indicate that viruses, not ambient pesticides, may be having a significant impact on the survivorship of eggs and larvae. This impact is so large that there are insufficient data on pupae or newly emerged adults to be able to make any statement whatsoever. If the preliminary results are confirmed as the remaining data analyses are completed, we will have a significant increase in the knowledge concerning the health of honey bee colonies. It may be possible to explain slow or non-existent colony build-up with our data. Second year colonies were used in all experiments conducted in 2009; second year rather than first year colonies were used to mimic conditions typically observed when miticides applications occur. First year colonies seldom need miticides treatment thus first year colonies were determined to not be representative of typical a typical honey bee colony exposed to miticides. The use of these second year colonies are the likely reason for poor performance (low to no egg hatch and no larvae survivorship) of the colonies as second year colonies have higher mite populations, mites are known vectors of several honey bee viruses, and a positive correlation between mite population and viral titers have been demonstrated by a number of experiments. A new avenue of research has opened up as a result of our observations and the work of Heather Matilla at Wellesley College. Dr. Matilla has found that the healthiest colonies are those whose queen has mated with a greater number of drones thus producing more sister families in a colony and that these sister families have different task specialties. We have noticed our weakest colonies, those with the highest viral titers, are the most likely to be under performing certain tasks. It is possible that some sister families are more susceptible to virus (or pesticide exposure) or conversely it may be that queens that are more poorly mate are more likely to have higher viral titers. The impact of miticides application on eggs, larvae and pupae survivorship and emerging adult longevity will continue next spring. To ensure low levels of virus in colonies, thus reducing/eliminating a confounding variable, all equipment will be irradiated this winter to eliminate any remaining viral contamination. New colonies (rather than second year colonies) will be used in all experiments. From our preliminary results an alteration of colony management techniques may be justified. The recommended changes will depend on the results of the data analysis to be completed.

Publications

  • No publications reported this period