Midnite Bee-Beekeeper's: Article ARTICLES An Evaluation of Menthol Placement in Hives of Honey Bees for the Control An Evaluation of Menthol Placement in Hives of Honey Bees for the Control of Acarapis woodi by E. W. HERBERT, JR.,* H. SHIMANUKI* and J. C. MATTHENIUS, JR.** ABSTRACT Menthol crystals were placed in various locations in hives of honey bees infested with the tracheal mite, Acarapis woodi. Samples of adult honey bees were collected every 3 weeks for 14 weeks and the tracheae were examined for the presence of mites. The presence or absence of living and dead mites was recorded. Administering the menthol by placing screen bags containing 50g (App. 2 oz.) on the top bars over the clustered bees appeared to be the best method of application in colder temperatures. Effective control did not begin until after the second treatment of menthol was applied on April 15. At that time temperatures exceeded 70'F, which seemed to be the minimum daytime temperature required for the vaporization of menthol. At the end of the study, all eight colonies were free of mites. INTRODUCTION THE HONEY bee tracheal mite, Acarapis woodi, was first discovered in the United States in 1984. Since then the mite has spread rapidly and it is now reported in 28 of the 48 contiguous states. At present, there is no chemical registered in the United States for the control of this mite, though several promising materials are currently being tested. Eischen et al. (1987) evaluated six compounds for control of the tracheal mite, and they found that Amitraz and bromopropylate showed reasonably good activity against adult mites.Unfortunately, neither showed much promise as a control for immature mites. The other Chemicals tested in this study gave little or no control of the mites. Amitraz gave the highest level of control of the six products tested. Another chemical being tested extensively for mite control is menthol. Menthol was first tested against A. woodi over 20 years ago in Italy (Giavarini and Giordani 1966). They tested 39 products in the laboratory for their effectiveness against Acarine disease. In the field, menthol produced the most effective control, was harmless to bees and did not contaminate the honey. In Texas, and Mexico, Wilson et al. (1987) tested menthol extensively for control of A. woodi, and they reported that the efficacy of menthol was excellent. Only small quantities or no menthol residues were detected in honey from treated colonies. In the present study, we evaluated the effectiveness of menthol(1) in colder climates. The tests were conducted using infested colonies of honey bees located near Vincentown, New Jersey, In addition, to determining the ideal time of year to begin treatment, we were also concerned with the placement of menthol within the colony. This had not been reported previously, since most studies were conducted in warmer climates. Under warm con ditions the vaporization of menthol is often accelerated, and-this could result in brood and adult bee mortality. However, in our New Jersey studies where the air temperatures in February and March are low, we had to develop methods to increase vaporiza tion. Consequently, we decided that the menthol should be placed over the winter cluster of bees instead of on the bottom board, as is often done in warmer climates. Since the vapors of menthol are heavier than air, they would tend to settle inside the colony. Also, placing the menthol over the cluster of bees would utilize the heat produced by the bees. MATERIALS AND METHODS Menthol was placed in various locations within the test colonies as follows: 1) Menthol (50g or App. 2 oz.) was enclosed in a 7 in. x 7 in. envelope made of plastic window screen (15 mesh/inch). Each screen bag was placed on the top bars of the combs directly over the winter cluster of bees. 2 2) Same as above, except the bottom entrance was taped shut and a small entrance in the top hive body was provided. 3) Four sponges (App. 7 in. x 4 in. x 2 in.) were each soaked in a 95 % ethyl alcohol - menthol solution and placed inside an empty hive body placed over the clustered bees. 4) A petri dish containing 50g of menthol was inverted over the clustered bees. 5) Two cloth bags, each containing 25g (App.1 oz.) of menthol, were hung between frames on either side of the clustered bees. 6) Untreated controls The 25 colonies were examined in January 1987 for infestation rates of mites before treatment and every 3 weeks thereafter (February 25, March 18, April 9, and May 7). The colonies were treated twice during the study; February 4 and April 15. Because of the cold temperature the menthol evaporated slowly, so the second treatment was not applied until most of the original menthol had evaporated. When collecting bee samples, we were able to detect menthol odors, even during February and March. Samples of 50 adult bees were collected and examined for A. woodi before treatment and every 3 weeks thereafter for a total of 14 weeks. The samples were collected by shaking a frame of bees into a small mailing tube (6 in. high x 2.5 in. diameter) using a funnel. Each tube of bees was immediately given a vial of sugar syrup which was replaced as needed until the bees were examined at the Beneficial Insects Laboratory, Beltsville, Maryland. The dissections were similar to those described by Eischen et al. (1986). The bees were first anesthetized with C02. The head and first pair of legs of each bee was removed with a scalpel by holdirig the bee on its back and gently pushing this section off with a downward, forward motion. The bee is then held between the thumb and forefinger for further examination using a dissecting microscope (40X). The first ring of the thorax was removed with a pair of fine forceps to expose the tracheal attachment to the thoracic wall, which is often the only place mites were found in a light infestation. Tracheae which appeared abnormal were removed with forceps, transferred to a microscope slide containing a thin film of glycerol, and gently dissected with a pair of fine needle probes. Living mites began to move immediately after the dissection. Mites were considered dead if they were discolored and did not move. This discoloration often ranged from light yellow to dark brown and dead mites often appeared to be desiccated and malformed. Living mites were translucent gray or pearl in color and in every case moved within a few seconds after dissection. The presence or absence of living and dead mites was recorded. When only eggs were present, the samples were scored as alive. RESULTS AND DISCUSSION Administering the menthol by placing screen bags containing 50g on the top bars over the clustered bees, appeared to be the best method of application in colder climates. We did not see any differences between colonies in which the bottom entrance was taped shut and those with the entrance open. On May 7, all eight colonies were free of mites. Effective control did not begin until after the second treatment of menthol was applied on April 15th. At that time daytime temperatures ranged from 30-79 degrees F. The menthol vaporization increased once daytime temperatures exceeded 70'F. This vaporization was probably accelerated by the placement of the menthol over the cluster. The test colonies had metal covered tops which probably intensified the vaporization process. Treating the colonies of bees with menthol soaked sponges also provided excellent control, and on May 7, these colonies were mite free. This method, although effective, requires more labor than the previously mentioned treatments. In warmer climates, 95 % ethyl alcohol- mentioned solutions, evaporate too rapidly resulting in bee mortality. Under such conditions, 70% ethyl alcohol is often used to slow evaporation. Alcohol is the solvent of choice, since menthol is very soluble in organic solvents such as alcohol, but only slightly soluble in water. In the present study, we were searching for methods to accelerate vaporization. Consequently, we used 95 % ethyl alcohol. Each sponge was soaked in the solution then placed in a plastic bag until it was placed in the test colony. Rubber gloves were worn during this procedure. Treating colonies by placing menthol (50g) in a petri dish lid which was inverted over the clustered bees did not result in complete mite control since on April 15 mites were still present. One problem with this method may have been the smaller surface area. In the treatments using the screen bag, or sponges, much of the area over the frames was covered. The petri dish, however, covered only a small area. Complete mite control was not achieved even by hanging two bags of menthol on either side of the clustered bees. In this treatment, the bees moved away from the menthol. We did not observe this in the other treatments, especially when the menthol was administered by placing it in the screen bags on the top bars. Two of the four control colonies died during the study. These colonies had pre- treatment infestation rates of 36 and 18%. Another colony, with an infestation rate of 36% at that time, was alive when the study was terminated on May 7. Interestingly, the control colonies contained the greatest mite populations in January and in every case the infestation level decreased during the study. This phenomenon was not observed in another study (Herbert et al. 1987) conducted in the same locatibn in New Jersey. In that study, the infestation rate in the control increased significantly during ihe study, often doubling in a 3-week period (from 30% to 66% in one instance). In the present study, the colonies were established from packagesIthe year before and all were relatively weak. Giavarini and Giordani (1966) reported that during the summer months the mite population declined even in the control colonies. Since the menthol treatments can be made in the winter when no nectar is available and the bees are consuming the winter stores, it is quite unlikely that honey stores will be contaminated. During the study, honey samples were collected and examined for menthol by Mr. Ed Kuta, Chemist, . New Jersey Department of Agriculture. The honey samples were "rippled" samples, that is, the samples were collected in the field by cutting out sections of sealed honey. These samples contained honey and beeswax. The honey and wax were separated and both were analyzed for menthol by high performance liquid chromatography. The menthol content of honey was highest (123.5 ppm) when the menthol was placed in cloth bags that were placed around the clustered bees. When the menthol was placed in screen bags (bottom shut), the menthol ranged from 3-46 ppm. When the entrance was open, the menthol levels were from trace-31 ppm. In the sponge treatment the menthol levels ranged from trace-37.9 ppm, and only traces were found when the menthol was placed in petri dishes, probably because of the smaller surface area. The controls contained 0.7 to 3.3 ppm menthol. Since menthol is a natural product obtained from peppermint or other mint oils, there is the possibility that menthol may be naturally present in honey. Field mint and peppermint (Mentha species) may contain from 2,850-24,000 ppm menthol in their foliage (J. A. Duke, personal communication, unpublished data). In summary, menthol was found to be an effective and safe treatment for colonies infested with A. woodi. Effective control began onice daytime temperatures exceeded 70'F. Placing the menthol (50g) in a screen bag directly on the top bars over the clustered bees resulted in the best control. Treatments could begin early in the season (March-April) in colder climates and then be discontinued before any major nectar flow to prevent the possibility of honey contamination. ACKNOWLEDGMENT We express; sincere appreciation to Walter Wilson and Paul Raybold, Senior Apiary Inspectom Edwin Kuta, Chemist; Mark Mayer, Entomologist, and Charles Wagg, Biologist, New Jersey Department of Agriculture, for their assistance in this study. LITERATURE CITED Eischen, F. A., J. S. Pettis, and A. Dietz. 1986. Prevention of Acarapis woodi infestation in queen honey bees with amitraz. Amer. Bee Jour. 126:498- 500. Eischen, F. A., J. S. Pettis, and A. Dietz. 1987. A rapid method of evaluating compounds for the control of Acarapiv woodi (Rennie). Amer. Bee Jour. 127:99- 101. Giavarini, 1. and G. Giordani. 1966. Study of acarine disease of honey bee. Final technical Report. Nat. Inst. of Apicult., Bologna, Italy. 34 pp. plus tables. Wilson, W. T., J. 0. Moffett, R. L. Cox, D. L. Maki, H. Richardson, and R. Rivera. 1987. Menthol treatment for Acarapis woodi control in Apis mellifera and the resulting residues in honey. proceedings International Conference on Africanized Honey Bees and Bee Mites. The Ohio State University, Columbus, Ohio (Inpress) . *Beneficial Insects Laboratory, U. S. Department of Agriculture, ARS, Beltsville, MD 20705 **N. J. Department of Agriculture, Trenton, NJ 08625 (1)Mention of a proprietary product does not constitute a recommendation by the USDA, nor does it imply registration under FIFRA as amended.