CONTROL OF THE 'VARROA MITE IN HONEYBEE COLONIES BY INTEGRATING CHEMOTHERAPY WITH CONVENTIONAL REQUEENING PRACTICE

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1 CONTROL OF THE 'VARROA MITE IN HONEYBEE COLONIES BY INTEGRATING CHEMOTHERAPY WITH CONVENTIONAL REQUEENING PRACTICE D,,'partment of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv Aviv 69978, Israel University, Ramat Summary A new method for controlling the mite Varroajacobsoni Oudemans in honeybee (Apis mellifera) colonies, which integrates chemotherapy with normal requeening practice, is described. By creating a 3-day gap in the presence of sealed brood all mites are simultaneously exposed to chemical treatment. Control using two fumigations with Taktic (amitraz 12'5%) approached 100%. The method has the advantages of efficiency, low cost, reduced chance of developing mite resistance to acaricide, and low health hazards for both consumer and beekeeper. Only 5.!r1O days of egg-laying are lost during confinement of the queen. Introduction The Varroa mite was first found in Israel during the summer of Levels of infestation in honeybee colonies were low and no influence on honey yield was noticed in the following spring. However by summer, after extraction of the citrus honey, levels of infestation had become high enough to exterminate a large number of colonies. Such severe damage was unexpected in the light of reports from Europe (Ritter, 1981) where it took as long as four years to cause serious damage to infested colonies. As a control measure the Israeli Ministry of Agriculture recommended fumigation with Taktic every three to four days for about four or five consecutive treatments. This succeeded in reducing the infestation level in many colonies and thus saved them from destruction, but even 20 consecutive treatments did not exterminate the mites. The reason for this was that the mites undergo their complete immature development inside the sealed brood cells where they are protected from the adverse effects of the acaricides. So many treatments are very costly and may hasten the development of resistance. The expense and th(; possibility of accumulating toxic chemicals in the honey raised dodbts about the future profitability of beekeeping in Israel. L: the present work, an alternative method for combating the Varroa mite was tested. In this met.od we produced a condition in the colony such that all mites were exposed simultaneously to the chemical treatment due to the absence of sealed cells. This method should be both cheap and practical and alleviate the disadvantages of multiple treatments. Materials and Methods The,;tudy was conducted with A. m. ligustica in standard Langstroth hives with one hive-body cont8.ining 10 frames. The chemicals used for mite control were formic acid (technical grade, 85%), in 25-ml doses that had been soaked in cotton on a plastic petri dish; or two drops of Taktic (which contains 12'5% amitraz) soaked on thick paper strips previously impregnated with 15% KN0 3. Above each hive-body was placed horizontally a wooden frame 5 cm high that accommodated the acaricide strip or dish on top of the frames within the hive. In order to be able to count the dead mites that had fallen from the combs it was necessary to prevent the bees from cleaning out the refuse from the bottom of the hives. For this purpose, we inserted, on the hive floor, a white masonite board on top of which a plastic screen was stretched at a height of 6 mm. Counting was conducted in the field with the aid of a head lens. Counted mites were removed one by one with forceps. Fumigation with Taktic served both as a means to control the mites in the hives, and as a check on their numbers. In order to determine whether Taktic was suitable for a population census, we conducted the following experiment. Seven colonies devoid of sealed brood were fumigated on each of three consecutive days and

2 the mites were counted on the masonite trays an hour after each fumigation. Prior fumigation, a sample of c. 300 worker bees was taken from each hive, placed in a 500 container with 6-mesh screened bottom mesh and immersed in a jar with 70% ethyl alcob The jars were shaken on a bottle shaker for 30 min to dislodge the mites as described by long et al. (1982), after which the alcohol was poured off and the mites counted (Table 1). comparison of numbers of mites dislodged after fumigation with numbers obtained by shakj worker bees in ethanol indicated that fumigation caused most, if not all, of the mites to di on to the trays (Table 1). Fumigation was therefore adopted as a means of estimating m population levels. TABLE I. Numbers of Varroa mites found on hive bottoms after fumigating honeybee colonies with Ta~ and by rinsing samples of worker bees in ethanol. Colony queens were caged. Before fumigation a sample of worker bees were taken from each colony a shaken with 70% ethanol to dislodge mites. The upper row of figures for each date gives the number of mi dislodged by the fumigation, the middle the average number of mites per 100 bees in the sample shaken w ethanol and the bottom row (parentheses) the number of bees in tlfe.ample. Days No. mites in colony after caging queen X SE !l '21 13'9 8'3 13"4 4'5 20'6 12'6 4'0!l'04 2'22 (329) (204) (149) (247) (277) (246) (225) I '71 0' " '06 0'06 (113) (116) (224) (255) (282) (279) (321) '57 0'65 0' '04 0'04 (395) (447) (269) (340) (249) (309) (213) < ~--o--~ FIG. 1. Sequence in normal requeening practice for honeybee colonies in Israel demonstrrting the overlap in sealed brood (stippled area) with introduction of the queen cell on the fifth day after removal of the old queen. a-removal of old queen; b-introduction of queen cell; c-beginning of egg laying.

3 FIG. 2. Sequence in a honeybee colony where the old queen is caged at day 1 and removed at day 10 when the queen cell is introduced. There is a 3-day period free of sealed brood (stippled). a-grafting for queen rearing in starter colony; b-caging old queen; c-introduction of queen cell and removal of caged queen; d-beginning of egg laying; e-inspection for egg-laying and fumigation; I-Repetition of fumigation. Requeening in Israel is usually conducted during the autumn, about October, when most beekeepers kill the old queen and replace her with a queen cell. This cell is usually introduced on the day of the old queen's removal or up to five days later. This procedure results in a cessation of oviposition for approximately 15 days (Fig. 1), however, there still remains an overlap of sealed brood between the progeny of the old and new queens. By preventing oviposition for five more days, this undesirable- overlap was avoided and a margin of safety of three additional days obtained during which all cells were unsealed thus rendering mites susceptible to fumigation. Instead of killing the queen, we caged her within the hive for 10 days, then removed her and introduced a new queen cell (Fig. 2). The queen cage was 4x 6 cm in size made of loosely-stapled window screen. It was suspended in the hive withoutattendants or candy. The sequence of operations for our main experiment was as follows: Day O. Larvae were grafted for rearing queens. Day 1. Queens in 22 colonies were confined in cages.' Day 10. The cage containing the old queen was removed from each hive and a queen cell introduced.. Day 24. Colonies were checked for egg-laying by new queens. Nine colonies (Group A) were treated with formic acid and 11 (Group B) with Taktic. At the time of treatrnent progeny from the new queens in the various colonies were as follows: five colonies had eggs only, 13 had eggs and larvae one day old, two had eggs and larvae one and two days old. Eight had a few old sealed cells containing live tanned pupae, which were removed. In two colonies there were 3 and 8 sealed cells, respectively, with larvae possibly from laying workers; these were uncapped. Day 27. The trays were removed and the mites counted, and colonies were examined for brood. All had unsealed brood abundantly provisioned. Sealed cells in a few hives were opened. All colonies were treated with Taktic. Day 42. Six colonies of Group A and five of Group B were treated with Taktic. Mites falling on the masonite trays were counted. Day 43. The same 11 colonies were treated again with Taktic and fallen mites were counted. Colonies were examined periodically throughout the experiment. Results At the time of removal of the caged queens on day 10 all were alive and active except for one which had died of injuries suffered during caging. In 21 colonies, a total of 16 queen cells (0-4)

4 TABLE 2. Numbers of VaTToa mites on hive bottoms following chemical treatment of colonies deprived of sealed brood. Colony queens were caged. Group A colonies were fumigated on day 24 (23 days after caging queen) with formic acid and on day 27 with Taktic (12'5% amitraz). Group B colonies were fumigated with Taktic on days 24 and 27. Dead mite counts were made one day after each treatment. Ml = mites killed by first treatment (formic acid for A, Taktic for B); M2 = mites killed by second treatment (Taktic); Ml% = kill by first treatment = Ml ~1 M2 x 100. The value of the G statistic for formic acid vs Taktic on day 24 = 161'6, P<O OOI. Colony no. Ml M2 Ml+M2 Ml% ' ' ' '0 5 IS ' ' ' ' ' i x 13'8 10'5 24' ' : ' ' ' ' ' ' ' ' '9 x 45'5 2'l 47'6 96 TARLE3. Numbers of VaTToa mites on hive bottoms following two treatments of honeybee colonies at onset of emergence of progeny of new queens. Treatments 1 and 2 were made on days 42 and 43 respectively from beginning of experiment. Group A had earlier received one fumigation with each of formic acid and Taktic in absence of sealed brood and Group B two fumigations with Taktic (see Table 2). Colony No. dead mites after no. Treatment 1 Treatment 2 Total Total x '91 1'18 3'10

5 per hive) were constructed despite the presence of a caged queen and in the 22nd where the queen had died there were 14. All these spontaneous queen cells were less advanced than those which had been introduced; all the latter would therefore have emerged first. On day 13 new queens were observed in the colonies and on day 22 new oviposition in all colonies except two, which were excluded from the experiment. The results of treatments on day 24 and d2 y 27 are listed in Table 2. The first treatment (day 24) with formic acid killed on the average only 57% of the total number of mites killed by the first and second treatment together (formic acid followed by Taktic). The first treatment with Taktic accounted for 96% of the total kill obtained with two treatments with Taktic. The difference l:jetween the effectiveness of formic acid and Taktic after the first treatment was highly significant statistically by the G test for independence (Sokal and Rohlf, 1969). The first of the new workers emerged on days 42 and 43. An average of three mites per colony were found (Table 3) indicating that either mite extermination was incomplete, or that the colony had been re-infested with mites from outside, or both. The chemotherapy caused no harm to the bees. All colonies in the experiment developed normally in the spring and produced normal honey yields from the citrus blossoms. No further treatment against Varroa was needed until the beginning of June, Discussion and Conclusions Our data show that it is possible in Israel to implement a management system for the Varroa mite that combines chemotherapy with the established practice of replacing the queen in the fall. The presence in the colonies of caged old queens, to a large degree, prevented the establishment of new queen cells. The few that were produced gave rise to queens that were younger than the queens from the introduced cells and did not constitute a problem. The desired condition of the presence of unsealed brood only, allowing the fumigants and vapours to penetrate to all the mites in the cells, was reached on the 25th to the 27th days of the experiments. The few sealed cells that were occasionally found could easily be uncapped during the examination for oviposition. Our system does not require any re-arrangement of combs in the hive or the removal and destruction of brood as suggested by Ruttner and Koeniger (1979). The lack of such treatments, and the high productive capacity of the new queens, coupled with the large supply of pollen accumulated in the oviposition-fre~ days, allowed for a rapid buildup of progeny from the new queens. In spite of the few mites found when the new workers emerged, control by our method, using two treatments with Taktic, was much better than. that obtained by beekeepers using more than 20 applications to colonies containing sealed brood. Our system, moreover, is inexpensive, requiring minimal extra labour and no special equipment. Formic acid, however, did not produce a high percentage kill; more research is recommended to enable its USt in the system. Although Taktic was effective, an acaricide that would pose less danger of honey contamination and less hazard to the beekeeper should be sought. A suitable schedule for the method is as follows:... Day O. Graft for queen rearing. Day 1. Check the number of accepted queen cells in the starter hive and cage an equivalent number of old queens. Day 10. Remove the cage with the old queen and introduce a queen cell. -Day ll~ Intwd-~en cell. Day 25. Check for egg-laying and fumigate with Taktic. If there has been no egg-laying, transfer a comb with eggs from another colony and, if available, a queen cell. Day 26. Repeat Taktic fumigation. Preliminary experiments indicated that the method is also adaptable to colonies where young laying queens are used for requeening. The new queen would be introduced on the 18th day when the old queen is removed. If a new queen is not available, the old queen can be released on day 20 and allowed to continue her egg-laying. This last variation can also be used in summer after the nectar flow if treatment for Varroa is needed before the time of requeening. Some tests were also made in which the caged old queen was taken off only after the young queen was laying (on day 25) so that the fertilization of the new queen was assured before the old one was killed.

6 The presence in our experiments of some mites following the second treatment emphasizes the problem of introducing newly-infested bees from outside the apiary. Hence it would be advantageous to treat all colonies at the site and even all of the apiaries in the area concurrently. Acknowledgements Thanks are due Dr. D. Eisikowitch of the Department of Botany of Tel-Aviv University for his encouragement and assistance; to Prof. David Wool for advice in statistical analysis; to Mr. J. Lubinjevski for technical assistance; to Ms. R. Suzin for the drawings and Ms. N. Paz for typing the manuscript. References DE JO~G, D., ANDREA-RoMA, D. DE; GONyALVES, L. S. (1982) A comparative analysis of shaking solutions for the detection of Varroa jacobsoni on adult honeybees. Apidologie 13 : RITTER, W. (1981) Varroa disease of the honeybee Apis mellifera. Bee Wld 62 : RCTT~ER, F.; KOENIGER. N. (1979) Experiments to eliminate Varroa mites by biological methods. 27 Int. Bcekeep. Congr., Athens: SOKAL, R. R.; ROHLF, F. J. (1969) Biometry. San Francisco CA USA: W. H. Freeman