Journal of Apicultural Science 109 AGE OF WORKER BEES PERFORMING HYGIENIC BEHAVIOUR IN A HONEYBEE COLONY B e a t a P a n a s i u k, W o j c i e c h S k o w r o n e k, M a ł g o r z a t a B i e ń k o w s k a, D a r i u s z G e r u l a, P a w e ł W ę g r z y n o w i c z Research Institute of Pomology and Floriculture, Apiculture Division, Kazimierka 2, 24-100 Puławy, Poland e-mail: beata.panasiuk@man.pulawy.pl Received 15 November 2010; Accepted 01 December 2010 S u m m a r y Hygienic behaviour of honey bees is the recognizing and removing of diseased or dead brood from the combs. The behaviour is a mechanism against brood diseases and limits the growth of parasite populations. The behaviour of individual bee workers, of different ages, in dealing with freeze-killed brood was observed in the beekeeping season of 2006. Observations were conducted in a single frame hive with glass walls. Shortly after emergence, young worker bees of carniolan race, GR1 line were marked with different shaped dorsal marks and put into the colony. Bee workers of different ages were involved in removing dead brood from cells, but the greatest activity was observed in the group of bees that were 6 days old at the beginning of the experiment. Some bees performed only certain tasks, while many of them participated in several activities. Keywords: hygienic behaviour, freeze-killed brood, age of worker bees. INTRODUCTION All the activities of worker bees in a colony are highly organized. The type of work performed mainly by individuals depends on their age, a phenomenon referred to as the "division of labour" (Rösch 1925, 1930, referred in Thompson, 1964; Lindauer, 1953; Thompson, 1964; Huang and Robinson, 1996; Arathi and Spivak, 2001). The type of work performed by individual workers of a particular age may vary, depending on the conditions inside and outside the colony (Momot and Rothenbühler, 1971; Robinson, 1992; Johnson, 2003). According to the same authors, each worker is able to perform all activities. The work activity also depends on the age of the bee. Some activities, however, require a specific physiological state of the worker bee. Hygienic behaviour is the ability of bees to detect diseased or dead brood in capped cells and remove it from the hive (Rothenbuler, 1964). The behaviour is genetically determined (Rothenbühler, 1964; Panasiuk et al., 2008; Borsuk, 2009; Bąk et al., 2010). Hygienic behaviour of bees depends on the strength and structure of colonies (Trump et al., 1967; Spivak and Gilliam, 1993), as well as weather and flow conditions (Thompson, 1964; Momot and Rothenbuhler, 1971; Dustmann and Ohe, 1998; Spivak et al., 1995; Jasiński et al., 2007; Spivak and Reuter, 1998; Panasiuk et al., 2009). The time it takes to recognize diseased or dead brood in cells is different. For this reason, the duration of the comb cleaning process is variable (Panasiuk et al., 2010). Observations of individual bee behaviour within the colony showed that only about 20% of workers are engaged in cleaning the cells. This amount is consistent with the proportion of young and old bees in the colony (Arathi and Spivak, 2001). Hygienic behaviour is most often performed by hive bees which are 14 to 20 days old. Hygienic behaviour is carried out in the period before foraging (Thompson, 1964; Momot and Rothenbühler, 1971; Arathi et al., 2000; Palacio et al., 2000; Arathi and Spivak, 2001).
110 However, cleaning is done by only a fraction of the individuals in the colony (Arathi et al., 2000; Boecking and Spivak, 1999). Bees which are 2 to 7 days old are probably too young to show an interest in dead brood removal (Spivak and Gilliam, 1993). According to Harbo (1995), 1 to 3 week old bees efficiently clean the combs. Workers younger than 4 days, as well as workers older than 25 days, do not participate in these activities. The first aim of our work was to determine the age of workers involved in the recognition of dead prepupe in capped cells and removing it. The second aim was to determine the specialization of bees in particular tasks. material and METHODS A small bee colony was set up in a single frame (370 x 223 mm in size), glass walled, observation hive, in July of 2006. A comb with brood at different developmental stages and with honey and bee bread was put into the hive. Then the hive was colonized with about 2 thousand bees of different ages and also a laying queen, to maintain the natural structure of the colony. In addition, the colony was fed with sugar syrup during the whole experiment. To obtain bees of known age, an instrumentally inseminated carniolan queen, GR1 line was closed in the single frame excluder, for one day, for egg laying. This was repeated four times at 5-day intervals. In this way, it was possible to obtain four groups of bees whose ages were known to differ by 5 days. Two hundred young bees from each age group, were marked shortly after emerging and added to the observation hive. The bees were differentiated with different shaped, numbered dorsal marks. On the day that the last group of bees were added to the hive, the worker bees were: 1-10 hours old and 5-, 10- and 15- days old. On the day the last group of bees were added to the experimental colony, the comb was replaced with another one with the younger brood. This was done so that the emerging bees did not increase the participation of young workers. The next day, in the central part of the comb, a small piece of a comb (5 cm to 7 cm) was inserted. This small piece of comb contained about 140 capped cells with freeze-killed brood (12 hours at -18 C) in prepupa stage. Before inserting the comb into the colony the piece was left for about 2 hours at room temperature to defrost. Immediately after that, a digital camera was set up to record the behaviour of bees. Each group of marked workers was composed of 200 individuals. Two sets of four different shaped dorsal marks, each set with numbers from 1 to 100 were used. The numbers of different shaped dorsal marks repeated twice. Only one group of bees was marked with one set of 100 white marks and one set of numbered marks (from 1 to 100) but the same shape. That was a group of bees which at the start of the observations were 6 days old. With such identification, it was possible to observe this group of bees and the participation of individual workers in particular activities. The behaviour of the bees was recorded using a digital video SONY DCR trv230e camera and infrared light. For bees, infrared light is synonymous with darkness. A recorded clear image covered an area with about 100 cells of dead brood. During the observation particular age-group of bees and individuals were noted to participate in following tasks: recognition which is detecting dead brood in capped cell, uncapping and cleaning cell interior. Recording began on July 15 th and ended 4 days later when all the cells had been cleaned. The film with the recorded cleaning process was watched later on a TV screen. The differences of number of "visits" of workers of known age, involved in cleaning activities were determined by Chi-square test (χ 2 ) using goodness of fit model for discrepancy between observed values and the values expected. For comparison of the participation of bees that were 6 days old at the beginning of experiment in particular cleaning activities the ANOVA Kruskal- Wallis test was applied.
Journal of Apicultural Science 111 RESULTS Age of bee workers involved in the removal of dead brood from comb cells Bee workers of known ages as well as unmarked bees participated in all stages of dead brood removal. Their participation in various stages of cleaning was different. Among the bees of known age, we observed 3138 "visits" associated with cell cleaning. Most of these bees were observed while they were cleaning the cell - 1286 times, and the least at the time of cell identification - 759 times. Bees that were 6 and 16 days old at the start of the observation were the age group significantly most often seen in the recognition of the cells (χ 2 = 10.27847, df = 3, p = 0.016343) (Tab. 1). They accounted for 35% and 31% of all marked bees that were found to be interested in Age of bees at the start of the observation and polished the cells the most frequently but significant differences were not stated (χ 2 = 2.523528, df = 3, p = 0.471055). Bees that were 6 days old at the start of observation participated the most in all activities related to cleaning the cells. In addition, the last column of Table 1 shows the total number of "visits" marked workers seen to be on the observed comb. This number includes both those who participated in the cells cleaning, as well as those that appeared on the observed part of comb, but were not involved in cleaning. Different numbers of workers were involved in particular stages of the cleaning process. Together 759 "visits", of marked bees of various ages, were observed in identification of a cell with dead prepupa. Each of the cells was "visited" at least 2 times. The highest number of "visits" was The number and percentage of "visits" of workers of known age, involved in activities related to cleaning cells with dead brood Bee activities Recognition the cell Uncapping the cell Cleaning the cell n % n % n % Number of "visits" of marked bees involved in cell cleaning All "visits" of marked bees observed - regardless of the activity 1 day 114 a 15.02 244 a 22.32 337 a 26.20 695 1333 6 days 263 b 34.65 322 a 29.47 398 a 30.95 983 1638 11 days 148 a 19.50 244 a 22.32 283 a 22.01 675 1435 16 days 234 b 30.83 283 a 25.89 268 a 20.84 785 1506 Total 759 100.0 1093 100.0 1286 100.0 3138 5912 n - number of "visits" Different letters indicate significant differences at p 0.05 T a b l e 1 a cell with dead brood. The youngest bees and the ones at the age of 11 days at the start of the observation participated less in these activities (15 and 19.5%, respectively). Bees of all age groups were involved in dead prepupe uncapping in similar percentages. Although 6- and 16-day olds bees were more frequently observed, however, significant differences were not confirmed (χ 2 = 1.405512, df = 3, p = 0.704243). The youngest workers, that were 1 day old (26%) and 6 days old (31%) at the beginning of observation, cleaned 20 times. The average was 7.5 times. When uncapping the cell an average of 10.7 "visits" were observed (the least 2 and the most 38). While in cleaning and polishing the cell a relatively higher number of bees were involved; on average 12.6 (4 to 35) "visits". Throughout the whole process of cell cleaning, a total of 3138 "visits" of marked workers were noted, with an average of 30.8 "visits" recorded in a single cell (the least 9, and the most 58). We counted the workers using the "stop/ hold camera position" and repeatedly
112 watching the cleaning process. This showed us that the marked bees were approximately ⅓ of the number of all the bees that were put into the experimental colony. Both marked bees, from 1 to 16 days old, and unmarked bees of unknown age, were involved in cleaning cells. Activities performed by particular workers in the process of removing dead brood from comb cells It was found that particular worker bees participated in a single cleaning activity, while others were involved in more activities throughout the whole cleaning process. Of the 100 workers with singlenumbered dorsal marks (that were 6 days old at the start of the experiment), only 80 individuals were seen in the cleaning process. They performed a total of 402 "visits". The remaining 20 bees were probably not accepted in the colony or died during the first days of life, or did not participate in the cleaning process. A lower number of the observed bees was involved in the recognition of a cell with dead prepupe. As many as 45 bees were seen to take part in that activity (56.2% of all bees from the group). They performed a total of 102 "visits" (each bee, on average 2.27 times). A larger number of bees participated in cleaning the interior of the cells. This work was performed by 63 bees (78.7%), and each of them "visited" a cell with dead prepupe an average of nearly 3 times. Despite of various involvement of workers in particular activities there were not statistically significant differences stated (Kruskal-Wallis Test: H (2, N= 161) =0.4348178, p =0.8046) (Tab. 2). Individual bees from the group were involved in cleaning in varying degrees. Fifteen workers were seen cleaning only once. Ten others were seen cleaning only twice, and the others from 3 to 10 times. Two attendants took part in cleaning as many as 15 and 16 times. One of the bees was observed 27 times: 4 times in the stage of the cell recognition, 8 timed during uncapping the cell and 15 times in the cleaning of the cell interior. Analysis of the activities performed by the same bee showed that 50% of the observed individuals participated only in a single activity: either in recognizing, uncapping or cleaning the cell. Remaining 50% of workers (40 individuals) participated in uncapping and cleaning the cell and more than half of these bees participated also in recognizing the cell (24 individuals) (Tab. 3). From the workers that were observed in the first stage of cleaning, only 4 did not participate in further activities, and the remaining 41 were observed several times in the remaining cleaning stages at different cells. It was found that 13 of the marked workers were involved only in uncapping the cell, and 23 only in cleaning the interior of cells (16.2 and 28.7% of all observed workers of the group respectively). The highest percentage of observed bees (50%) were involved in uncapping and cleaning the interior of cells (Tab. 3). Bee workers which were 6 days old at the beginning of observation involved in subsequent cells cleaning activities T a b l e 2 Cleaning process activities Recognition of cell Number of bees involved in the cleaning activities Number of "visits" of bees Number of bees Particular activities Average "visits" per bee % of bees 102 45 2.27 a 56.25 Uncapping the cell 80 123 53 2.32 a 66.25 Cleaning the cell 177 63 2.81 a 78.70 Total 80 402 161 2.49 100 The same letters indicate that differences are not significant
Journal of Apicultural Science 113 T a b l e 3 Number of workers and activity performed by individual bees (specialization) Bee activity Number of bees involved Bees involved in a particular activity in cleaning activities number % Recognition of cell 4 5.0 Uncapping the cell 13 16.25 Cleaning the cell 80 23 28.75 Uncapping and cleaning the cell 40 50.0 - among them also in recognizing the cell 24 30.0 DISCUSSION The activities that were components of the bee hygienic behaviour, were recognition of dead brood in capped cell, uncapping and cleaning the cell (Panasiuk et al., 2010). In addition to these activities, we observed other bee behaviours as was noted in the studies of Arathi and Spivak (2001). Different bees, marked and unmarked, walked on the comb, checked cells, auto-groomed or flew away from the hive, and some brought back pollen or nectar. In our experiment it was shown that worker bees of different ages take part in cleaning dead brood from cells. However, the bees that were 6 days old at the beginning of our observation were the most often involved in these activities. This confirms reports by Harbo (1995), who observed that workers younger than 4 days, and older than 25 days, did not take part in cleaning and that hygienic behaviour was exhibited by 1 to 3 weeks old workers. Experiments conducted by Thompson (1964) and Arathi et al. (2000) showed that the hygienic behaviour is performed mainly by bees 27 days old or younger. Older individuals are also involved in cell cleaning, especially at the period of strong food flow (Thompson, 1964; Rothenbühler and Momot, 1971). A small number of young marked bees observed on the hive entrance during these observations confirm that worker bees remain in the hive during the first two weeks of life. In described observations from the group of 100 single numbered bees which were 6 days old at the beginning of observation, only 80 were seen in the experimental colony. The remaining 20 were either not accepted in the colony, died, or did not participate in the cleaning process. It can be assumed, that the number of observed worker bees of other age groups in relation to the initial number of bees added into the hive, was the same or similar. Our observations of individual bee behaviour, on the basis of workers from the group with single numbers on dorsal marks, showed that 50% of the bees performed only a single activity. The second half was involved in more activities: all in uncapping and cleaning the cells and many of them also in recognizing. These results are confirmed by the data obtained in earlier studies of other authors (Arathi et al., 2000; Arathi and Spivak, 2001). According to them, the individual bees may participate in different activities: detecting cells with diseased or dead brood, uncapping and cleaning them, but rarely bees clean the cells with damaged brood from the time of cell recognition until the cell was completely cleaned. CONCLUSIONS 1. In the process of cell cleaning, worker bees of different ages are involved, but the most frequent are 6 (to 10) and 16 (to 20) days old bees. Only in cleaning the cell the 1 and 6 days old bees were predominately. 2. Individual bee workers participate in various activities related to cleaning the cells but some of them specialize in only a single activity.
114 REFERENCES Arathi H. S., Burns I., Spivak M. (2000) - Ethology of Hygienic Behaviour in the Honey Bee Apis mellifera L. (Hymenoptera: Apidae): Behavioural repertoire of Hygienic bees. Ethology, 106(4): 365-379. Arathi H. S., Spivak M. (2001) - Influence of colony genotypic composition on performance of hygienic behaviour in the honeybee, Apis mellifera L. Anim. Behav., 62: 57-66. Bąk B., Wilde J., Siuda M. (2010) - Comparison of hygienic behaviour between five honey bee breeding lines. J. Apic. Sci., 54(2): 17-24. Boecking O., Spivak M. (1999) - Behavioural defenses of honey bees against Varroa jacobsoni Oud. Apidologie, 30(2-3): 141-158. Borsuk G. (2009) - Influence of increased division of labour among worker bees on dead brood removal. J. Apic. Sci., 53(2): 69-77. Dustmann J. H., Ohe W. von der. (1998) - Einfluβ von Kälteeinbrüchen auf die Frühjahresentwicklung von Bienenvölkern (Apis mellifera L.). Apidologie, 19(3): 245-254. Harbo J. R. (1995) - Observations on hygienic behaviour and resistance to chalkbrood. Am. Bee J., 135(12): 828. Huang Z-Y., Robinson G. E. (1996) - Regulation of honey bee division of labor by colony age demography. Behav. Ecol. Sociobiol., 39: 147-158. Jasiński Z., Madras-Majewska B., Gruszczyńska A. (2007) - Dynamika czyszczenia komórek z martwego czerwiu a produkcyjność rodzin pszczelich. Materiały z XLIV Naukowej Konferencji Pszczelarskiej, Puławy, 24 25 kwiecień 2007: 8-9. Johnson B. R. (2003) - Organization of work in the honeybee: a compromise between division of labour and behavioural flexibility. Proc. R. Soc., 270: 147-152. Lindauer M. (1953) - Division of labour in the honey bee colony. Bee World, 34: 63-73. Momot J. P., Rothenbuhler W. C. (1971) - Behaviour genetics of nest cleaning in honeybees. VI. Interactions of age and genotype of bees, and nectar flow. J. Apic. Res., 10(1): 11-21. Palacio M. A., Figini E. E., Ruffinengo S. R., Rodriguez E. M., Del Hoyo M. L., Bedascarrasbure E. L. (2000) - Changes in population of Apis mellifera L. selected for hygienic behaviour and its relation to disease tolerance. Apidologie, 31(4): 471-478. Panasiuk B., Skowronek W., Bieńkowska M. (2008) - Influence of genotype and method of brood killing on brood removal rate in honey bee. J. Apic. Sci., 52(2): 55-65. Panasiuk B., Skowronek W., Gerula D. (2009) - Effect of period of the season and environmental conditions on rate of cleaning cells with dead brood. J. Apic. Sci., 53(1): 95-103. Panasiuk B., Skowronek W., Bieńkowska M., Gerula D. (2010) - Process of clearing dead brood from cells in a honeybee colony. J. Apic. Sci., 54(1): 5-11. Robinson G. E. (1992) - Regulation of division of labour in insect societies. Ann. Rev. Ent., 37: 637-665. Rothenbuhler W. C. (1964) - Behaviour genetics of nest cleaning in honeybees. I. Responses of four inbred lines to disease killed brood. Anim. Behav., 12: 578-583. Spivak M., Gilliam M. (1993) - Facultative expression of hygienic behaviour of honey bees in relation to disease resistance. J. Apic. Res., 32(3-4): 147-157. Spivak M., Reuter G.S., Lamb M. (1995) - Frequency of hygienic behavior in naturally mated daughters of a hygienic breeder. Am. Bee J., 135(12): 830. Spivak M., Reuter G. S. (1998) - Performance of hygienic honey bee colonies in a commercial apiary. Apidologie, 29(3): 291-302.
Journal of Apicultural Science 115 Thompson V. C. (1964) - Behaviour genetics of nest cleaning in honeybees. III. Effect of age of bees of a resistant line on their response to disease-killed brood. J. Apic. Res., 3(1): 25-30. Trump R. F., Thompson V. C., Rothenbuhler W. C. (1967) - Behavior genetics of nest cleaning in honey bees. V. Effect of previous experience and composition of mixed colonies on response to disease-killed brood. J. Apic. Res., 6(3): 127-131. WIEK ROBOTNIC PRZEJAWIAJĄCYCH ZACHOWANIE HIGIENICZNE W RODZINIE PSZCZELEJ P a n a s i u k B., S k o w r o n e k W., B i e ń k o w s k a M., G e r u l a D., W ę g r z y n o w i c z P. S t r e s z c z e n i e Rozpoznawanie i otwieranie komórek z chorym lub martwym czerwiem oraz usuwanie go z komórek plastra określane jest zachowaniem higienicznym. Jest ono mechanizmem hamującym rozprzestrzenianie się chorób lub pasożytów w rodzinie pszczelej. W 2006 roku obserwowano zachowanie robotnic w różnym wieku w stosunku do czerwiu zabitego przez mrożenie. Obserwacje prowadzono w jedno-ramkowym ulu obserwacyjnym ze szklanymi ścianami. Młode robotnice pochodzące od matki rasy kraińskiej linii GR1 tuż po wygryzieniu z komórek znakowano różnokształtnymi opalitkami i dodawano do rodziny doświadczalnej. W oczyszczaniu komórek brały udział robotnice w różnym wieku, jednak największe zaangażowanie przejawiały pszczoły w drugim tygodniu życia. W identyfikacji komórki z martwą przedpoczwarką najliczniej brały udział robotnice z grup, które w dniu rozpoczęcia obserwacji były w wieku 6 i 16 dni (odpowiednio 35 i 31%), natomiast mniej licznie pszczoły jedno- i jedenastodniowe (15 i 19,5%). Z wyjątkiem pszczół sześciodniowych, które przeważały, udział pozostałych grup w odkrywaniu komórek był zbliżony, natomiast w oczyszczaniu wnętrza komórek dominowały robotnice dwu najmłodszych grup: 1-no dniowe (26%) oraz 6-cio dniowe (31%). Połowa z pośród pszczół sześciodniowych wykazywały ścisłą specjalizację wykonując tylko wybrane czynności, natomiast pozostałe uczestniczyły w różnych pracach. Dwie czynności: odkrywanie i oczyszczanie wnętrza komórek wykonywało 50% obserwowanych pszczół, wśród których ponad połowa (ale 30% całej grupy pszczół) było zaangażowanych również w identyfikację komórki. Słowa kluczowe: zachowanie higieniczne, czerw mrożony, wiek robotnic.