Progress 09/01/04 to 08/31/08
Outputs
OUTPUTS: The aim of this project was to develop an effective treatment for the Varroa mite, a parasite of honey bees that is decimating colony populations throughout the world. We used a proprietary sulfur formulation that provides, for the first time, stabilized colloidal sulfur. The first half of Phase II research focused primarily on identifying an appropriate delivery system that could be scaled up for wide distribution and testing it in the laboratory. The second half of the project focused on field testing the system, performing toxicity testing, redefining formulations and preparing data for registration. Initial candidate delivery substrates focused on filter-type materials. While promising, these proved too hard for bees to shred and distribute throughout the colony effectively, and they sequestered too much of the treatment. We found treatment surface area was correlated with efficacy with larger surface areas producing higher mite mortality rates. Spraying maximized surface area, and results in laboratory feeder-cage studies showed 50 - 77% mite drop compared to 24% for the control. There was no evidence of bee toxicity, even at levels 10 fold higher than the prescribed dose. Toxicity testing showed that PRC colloidal sulfur was the least toxic of all the sulfur formulations tested. This included lime sulfur and elemental sulfur. Surprisingly, while initially benign, elemental sulfur showed a profound toxicity after 72 hours. Field tests in 2005 used the spray application method to test two colloidal sulfur formulations. Results were very good, with an average 52% mite drop compared to 26% for the control over a three-day treatment period. Subsequent field tests in 2006 showed equivocal results. In some cases the treatment increased mite drop. In others it reduced mite drop. Both results could be the consequence of a deleterious effect on mite load (increased drop) and mite reproduction (decreased drop.) The number of reproductive mites per cell did not correlate with treatment. Limited resources prevented additional analyses of this kind. To expedite registration, we tried a different raw material in the synthesis of the colloidal sulfur that already has EPA registry and toxicology data available. While successful synthetically, the properties were not as good as the original formulation and shelf life was poor. We conducted experiments that demonstrated the colloidal sulfur is the active ingredient in the treatment and that raw materials are inert. In this regard, the lab mite bioassay gave somewhat equivocal results while the cage study gave a clear demonstration that the raw material is inert. A deficit in CRADA partner resources (mites, bee colonies, partner beekeepers) throughout this study impaired our ability to execute certain planned experiments and made others impossible to repeat. We addressed this in part by purchasing and establishing 25 of our own colonies. In a surprise development, we observed that the colloidal sulfur treatment significantly reduced the viral load in diseased honey bee colonies. PARTICIPANTS: This project was conducted under the direction Dr. Judith Hooper of Pima Research Company. Expert technical assistance was provided by Maria Williams, Nicole Barrentine, Kari Seward, and Brian Chaszar of PRC. Maria Williams and Brian Chaszar were college students who were able to use their experience with the project to acquire new laboratory skills and gain experience in experimental design and data interpretation. Ms. Williams and Ms. Barrentine have both been accepted into PhD programs. Mr Chaszer plans to go on to graduate school after receiving his BS in winter 2008. We wish to acknowledge the generous support of the USDA/ARS Carl Hayden Honey Bee Research Center (Tucson, AZ) who provided specialized research facilities for the study of honey bees under a Cooperative Research and Development Agreement (CRADA.) We are particularly grateful to the following people from the Research Center: Dr. Gloria Degrandi-Hoffman who showed broadminded interest in our project, offered a warm welcome to her facility, and generously shared very limited resources and offered lively discussion; research scientist Dr. Diana Sammataro and research technician Jennifer Finley provided guidance in honey bee management, experimental design, and the interpretation of data relating to varroa infestation; research technician Tom Deeby gave meticulous attention to the details of honey bee colony management. Dr. Gordon Wardell of SAFE Research and Development (Tucson, AZ) graciously shared insights and experimental design strategies particularly those relating to varroa infestation of honey bees. Our Phase III partner was Ag Formulators (Fresno, CA.), specifically Don Holbrook, Shawn Holbrook and Chris Louie. Besides generously supplying raw materials, they proved enthusiastic partners, kept us focused on necessary practicalities, and helped create a coherent approach for successfully registering, manufacturing, and marketing the finished product. TARGET AUDIENCES: The primary target for this research is the professional beekeeping industry, but also includes hobbyists. The secondary target for this research is other researchers and scientists working in the area of varroa mite control, colony collapse disorder. It is also targeted to those interested in promoting non-toxic pesticides. PROJECT MODIFICATIONS: The major change in the research program was the full year delay (or more) in product field testing and the limited laboratory and in-house testing we were able to perform because of the lack of resources -- in particular, bees -- supplied by our CRADA partner. This was exacerbated by the reluctance of commercial bee keepers to field test the product due to a very strong market for bee hives to be used in pollination. This later turned into a reluctance due to spreading disease (colony collapse disorder) and concern over losing more hives should field tests fail. The availability of mite infested hives for testing remained a serious and constant problem.
Impacts
The use of sulfur for the control of the varroa mite is a sound idea with promise and potential. We saw that the formulation is non-toxic and was easy to handle, and field application was straightforward and uncomplicated. We learned from our Phase III partner that the economics of production are very attractive with small investments needed in raw materials and labor. We discovered that distribution would be uncomplicated thanks to existing cooperative type dealerships that are the primary supply source for both commercial and hobby beekeepers. Based on initial data for registration, and feedback from our Phase III partner's legal counsel, it appears obtaining a label for use would be uncomplicated and could be expedited if we could change certain raw materials or if we could demonstrate mode of action was due solely to the colloidal sulfur. We were not able to achieve the former, but did unravel the latter. The field trial results were inconclusive. with no clear trend in mite mortality that we could detect using our measurement strategies. We observed that, besides the inherent difficulty in comparing colonies that are not identical, variability in results might also reflect the appearance of widespread disease and colony collapse disorder characteristic of so many colonies during the 2005-2007 seasons. Accordingly, we think more field-testing is in order before going forward with commercialization and expect it will be required for registration. But to our mind, the most compelling and interesting result of the study is the indication that the proprietary colloidal sulfur treatment can mitigate viral infestation of honey bee colonies. To the extent that this result is due to some kind of interference with mites in their capacity to act as vectors for these viral diseases, this is an extremely important discovery within the scope of this SBIR Phase II project. We were profoundly disappointed to learn that our Phase III partner was bought out by a competitor company in late 2007, and the new arrangement precluded a continued relationship with Pima Research Company. We hope to move forward with introducing the colloidal sulfur treatment to the marketplace with a new Phase III partner. We also hope to explore the viral mitigating effects further under another proposal and, again, with a new Phase III partner.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
The goal of the project is to develop an efficacious method for using sulfur as a treatment for varroa mite infestation of honey bees. Outputs consisted of 1) developing prototype application methods that could be easily adopted by beekeepers; 2) conducting treatment field trials using the prototype application technique; 3) evaluating the field tested sulfur treatment on colony health; 4) further assess the potential for toxicity due to over-application. A variety of potential application methods were considered that would be easy for beekeepers to use in the field and, at the same time, would be easy and inexpensive to manufacture. Five field trials were conducted. Four used honey bees owned and managed by professional beekeepers from around the US, while the fifth used honey bees owned by Pima Research Company and managed by USDA / ARS Honey Bee Research Laboratory personnel at the USDA / ARS facility in Tucson. The efficacy of the treatment as a mite treatment was
determined by counting the mites captured on sticky boards placed on the bottom of the hive over a fixed period of time. Appropriate data collection strategies were developed that beekeepers could easily follow and would not abandon due to complexity or excessive time commitment. The effect of sulfur treatment on colony health was assessed. Brood count and colony strength was estimated and any adverse affects on bee behavior or queen laying was noted. Additionally, we sought to measure whether the presence of the sulfur treatment might affect the varro mite mediated transmission of honey bee viruses. Judy Chen Ph.D. at the Beltsville USDA facility, provided her expertise in this area. Samples of sulfur treated and untreated bees from the field trial at the Tucson facility were collected and sent to Dr. Chen according to her protocols. Using PCR techniques, the samples were evaluated for the presence of 6 pathogenic viruses known to compromise honey bee colony health. A sample size of
50 bees per colony and a minimum of three colonies per test group were taken. The data were organized to compared efficacy of the sulfur treatment as a function of particle size, and as compared to a control group with no treatment. The effect of raw material choice and synthesis scheme on the toxicity of the final colloidal sulfur formulation was evaluated. These tests were performed on live bees in cages over a 5-day period. These results reflected a higher dose of application and a longer evaluation period, and were combined with earlier data where testing was done at a lower dose. Additionally, the shelf-life and stability of the colloidal sulfur product as a function of raw material choice and synthetic scheme was evaluated.
Impacts
Keen to utilize equipment and techniques already familiar to beekeepers, we learned we could use a spray application method that employed commonly existing spray equipment if brass parts were replaced with plastic or Teflon. The replacement was necessary as the sulfur compound will corrode metal parts. We found it easiest to supply the treatment as a concentrated colloidal sulfur preparation pre-measured in plastic tubes. Beekeepers reported the prototype application method was straightforward to easy to use. They also reported the directions for data collection were easy to understand. The efficacy of the treatment applied in field trials was determined by counting the mites captured on sticky boards placed on the bottom of the hive over a fixed period of time. These data were analyzed using a variety of statistical technique to eliminate the widely variable background in all field trials data sets. We found the raw data was chaotic due to the effect of gross
differences in colony population, the percent drones population per colony, African bee invasion, and disease. In order to minimize these differences between colonies, post treatment mite drop data was normalized to pretreatment values. With the data thus normalized, and the data graphed as box plots, trends were observable that differentiated treatments from the control. Evaluated using ANOVA, these data failed the Tukey test for significance. We observed that the treatment had no adverse affects on colony health. Indeed, it was noted that the treated colonies the controls. While the varroa mite is known to destroy honey bee colonies through feeding on bee larva (and thus removing the next generation of honey bee), its devastating effects are also due to vectoring viruses. Went bees were analyzed for the presence of these viruses, we learned that the sulfur treatment reduced viral load dramatically. We noted that antiviral activity increased with decreasing sulfur particle size. A
second series of toxicity tests were performed. We discovered there was significant toxicity at 6 times the recommended concentration (67 percent mortality) and at 8 times (92 percent mortality). Importantly, we learned that a powdered sulfur treatment (applied as a positive control) killed 95 percent of the bees by the fourth day of the test even though there was no evidence of any toxicity initially. This last finding is especially important as beekeepers are likely to use readily available "flowers of sulfur" from their local Farmers Coop and this could produce a deadly result. It also indicates that the colloidal sulfur preparation is a safer alternative, giving us a marketing edge and positioning the product a bit more uniquely.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
The goal of this project is to develop a treatment for Varroa mite infestation of honey bees that does not select for resistant strains and that does not pose the toxicity and handling problems that are inherent with the types of pesticides currently in use. Phase I research showed that our novel sulfur meet environmental concerns and resistance issues, while demonstrating good Varroa control in the lab. Early stages of Phase II field-testing showed that this formulation reliably produced 50 - 70% mite drop based on total mite load. We felt this might be improved with better application methods and/or modified treatment formulation. Application method: Our initial idea to mobilize the treatment on fiber strips failed. This method relied upon bees to shred and distribute the treatment-coated fibers, but success depended too much on the highly variable [hygienic] behavior of bees. Our CRADA partner suggested pouring the treatment between frames of bees. This technique
also failed as it relied too heavily upon pouring the treatment evenly and directly on the frames and not through the space between the frames. Again, distribution was inconsistent. We next tested spraying the treatment between frames of bees using a pressurized or handheld sprayer. We determined that a plastic or Teflon nozzle was preferable. Spraying was straightforward and uncomplicated; did not depend upon bee behavior or too heavily upon applicator technique to get a consistent application. As it is an easy, familiar, and cost effective technique for beekeepers, we continued with this technique for subsequent field-testing. Field testing: Testing during fall of FY 2006 was somewhat confounded by demand for commercial honey bees for pollination, and spring testing was delayed due to wet weather. Testing done on site with small nucleus colonies and young (1 year old) 10 frame colonies indicated that the sulfur formulation continued to show moderate effect, on average, with large
variations between colonies ranging from no apparent mite drop to a dramatic and near complete mite drop. We ascribe some of the variability to application technique and some to differences in colony genetics, strength, and behavior. In addition to mite counts, we are also evaluating treated colonies for changes in brood laying, bee behavior, and evidence for disease. Neither we, nor our collaborators, have observed any adverse side effects of the treatment. Formulation modification: We learned that both our CRADA partner and beekeepers alike have a perceptual bias that good mite control requires immediate mite drop. Thus, with the aim of improving immediate mite-drop performance, we developed two modifications of our sulfur formulation to test alongside a simple increase in the treatment concentration. The new formulations were also tested for shelf life where we looked for changes in particle size that might affect performance, and the appearance of microbial growth. One of the new
formulations failed shelf life testing when it developed considerable bacterial growth after three months storage. The remaining two have been included in FY 2007 field-testing currently underway.
Impacts
The commercial availability of an effective treatment for controlling the present widespread infestation of honey bee colonies with Varroa destructor is crucial to preserving the integrity and strength of the US agricultural economy. The importance of honey bees to U.S. agriculture is immense; by improving quality and yield, honey bees add over $14.6 billion to crop value. Varroa infestation has produced large losses in the managed bees of commercial operations. Small rural operations are particularly hard hit. The number of total beekeepers has declined, and there is an unwanted downsizing of remaining operations. The result for growers has been decreased have availability that are now more expensive to lease. The current resistance of the mites to pesticide treatment points to a greater urgency for developing alternative treatments, especially as beekeepers turn to highly toxic and often illegal treatments. The current research supported by this SBIR grant will
generate a treatment that can abate the debilitating infestation of Varroa using a special sulfur formulation that is environmentally safe to use. Besides protecting the beekeeping and agricultural industries, treatment and control of Varroa with this product will help preserve the natural ecologies and other environmental resources that depend on pollination for reproductive success and plant health. The elimination of the toxic, and mostly ineffective, pesticides currently employed to combat Varroa will insure that honey and other hive products maintain their status as 'natural' and safe consumables.
Publications
- No publications reported this period
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