PROCESS OF CLEANING DEAD BROOD FROM CELLS IN A HONEYBEE COLONY

Journal of Apicultural Science 5 PROCESS OF CLEANING DEAD BROOD FROM CELLS 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 Research Institute of Pomology and Floriculture, Apiculture Division Kazimierska 2, 24-100 Puławy, Poland e-mail: beata.panasiuk@man.pulawy.pl Recieived 04 November 2009; Accepted 25 May 2010 S u m m a r y Hygienic behaviour of honeybees is described as recognizing, and removing diseased or dead brood from capped cells. This behaviour is a mechanism of honeybee resistance against the brood diseases. It also limits the growth of the parasitic mite population. The process of dead brood removal was observed in 2006. An experimental colony was set up in a 1-frame glass hive. A piece of comb containing 102 capped cells with freeze-killed brood was observed. The cleaning cell process was divided into successive stages, i.e.: recognition of cell with dead brood, opening with mandibles the wax capping, removal of dead brood, cleaning and polishing the cell. It was noticed that the queen laid eggs in some of the cells that had been cleaned by workers. The successive stages of cell cleaning started and finished at various time periods. The time to clean the cell differed as well. Bees cleaned all the cells within 3 to 85 hours, on the average during 46 hours. Keywords: hygienic behaviour, freeze-killed brood, cleaning stages. INTRODUCTION Dead brood, regardless of the reason of its death, is always recognized in capped cells and removed from the comb by worker bees. This is one of mechanisms of honeybee resistance against brood diseases (Apis mellifera) and it was called hygienic behaviour by Rothenbuhler (1964). The efficiency of the dead-brood removal depends on the genetic factors (Rothenbuhler, 1964; Heath, 1982; Spivak and Gilliam, 1993; Büchler, 1996; Spivak and Reuter, 1998; Arathi et al., 2006; Panasiuk et al., 2008), a colony strength and structure (Trump et al., 1967; Mattila and Otis, 2006) and on the manner of the brood killing (Békési and Szalai, 2003; Bieńkowska et al., 2004; Panasiuk et al., 2008). It has been also shown that weather (Dustmann and Ohe, 1998; Jasiński et al., 2007; Panasiuk et al., 2009) and foraging conditions (Thompson, 1964; Momot and Rothenbuhler, 1971; Spivak et al., 1995; Spivak and Reuter, 1998; Panasiuk et al., 2009) influenced expression of the hygienic behaviour. Woodrow (1941) showed that the time required to clean out the cells was proportional to the amount of diseased or dead brood in a given colony. Others have stated that it takes hygienic bees the same amount of time to remove a similar number (from 100 to 1000) of killed larvae or prepupae, regardless of the number of given cells or the stage or development (Jones and Rothenbuhler, 1964). Studies performed in small experimental colonies shown that worker bees did not clean out all diseased or dead brood

6 (Spivak and Gilliam, 1993). Hygienic colonies of medium strength removed 95% of freeze-killed brood within 48 hours, while weakened ones only 75%. Nonhygienic colonies removed 34 and 26% of dead brood, respectively. Many researchers observed the behaviour of bees while workers were removing dead brood from the comb cells (Robinson, 1987; Arathi and Spivak, 2001). They also tried to estimate the time, which was necessary for a single cell cleaning (Robinson, 1987). However, the complete process of cells cleaning in a honeybee colony has not been described so far in details. The aim of the present research was to know the behaviour of bees during the dead brood removing. It was decided, to determine time of bee reaction to dead brood in capped cells, as well as the sequence and lasting time of the successive stages of the cleaning process. MATERIAL AND METHODS An experimental bee colony of the carniolan race, GR1 line, was set up in a single frame hive with the glass walls in July of 2006. The hive was colonized with about 2 thousand bees of different ages, as well as, with an egg laying queen. A comb with brood at all the developmental stages, and with honey and pollen was introduced into the hive. A piece of a comb (5 x 7 centimeters) with brood at the prepupa stage (12 to 13 days old) from another colony was taken. The piece, which contained about 140 capped cells on each side, was frozen for 12 hours at a temperature of -18 C. Before inserting it into the experimental colony the piece was left in a room temperature to unfreeze. The colony was fed with sugar syrup during the whole observation period. The cleaning behaviour of bees was recorded with a SONY dcr trv230e digital camera under the infra red light conditions in order to not disturb the bees. A recorded clear image focused on the comb area with 102 cells. Recording started on July the 15 th immediately after inserting the piece of comb into the experimental colony, and ended after 4 days, when all the observed cells were cleaned and 96 hour of film was made. The film with the recorded cleaning process was watched later on the 28 TV screen. Transparent foil with marked cells with freeze-killed brood was attached to the screen to facilitate identification and observation of particular cells. The stages, the average time of these stages, and the time of the complete cell cleaning process were determined. The moment of egg lying by the queen was noticed in some of the cleaned cells as well. RESULTS The following, successive stages of the cell cleaning process were identified: - time before identification - recognition - first symptoms of interest and recognition of the cell - uncapping - opening wax capping with mandibles - cleaning - removing of prepupae and polishing the cell and - egg laying by the queen. Some of the stages of cell clearing (Fig. 1) were easily determined, whereas others were difficult strictly to define. It was not difficult to determine the time of the uncapping the cell, by noticing a small hole appearing on it, or the time of egg lying. It was more difficult to define the first interest with a cell as the moment of recognizing a dead brood inside, the ending of wax uncapping, and the starting of dead brood removal. The boundary for prepupae removal and polishing the cells were impossible to define. For this reason both stages were pooled together. The first reaction of bees to dead brood in some of the cells and its identification was observed just few minutes after the experiment had started (Fig. 1). In other cells it took several hours after the piece of comb with freeze-killed brood had been inserted into experimental colony, for it to be recognized. The whole cleaning process in some of the cells was completed in a short time, e.g. in cell No. 3 the first two

Journal of Apicultural Science 7 Fig. 1. Successive stages of bees activity during the dead-brood removal. Each of the cells was treated separately

8 stages (recognition and cell uncapping) were completed in 20 minutes. In cell No. 3 the whole cleaning process was completed in about 3 hours. The longest cleaning process of 85 hours was observed in cell No. 10. The whole cleaning process in most of the cells was completed within 50 hours of the observation. In the other twenty cells the process took about 70 hours while in cell No. 10 it was completed in 88 hours. Once the piece of comb with freeze-killed brood was inserted into the colony it took various periods of time for the bees to show an interest in it. It took from 5 minutes in cell No. 89 up to 14 hours 40 minutes in cell No. 93 (Tab. 1, Fig. 1). On an average it took 2 hours and 55 minutes. The first interest was recognized as the time when a bee momentarily stayed on a certain cell capping and drags it with its mandibles. That stage was considered to have ended when a hole had appeared on it. The earliest show of interest was noticed five minutes after beginning of observation when bees started dragging the wax capping in cell No. 89. The average for this stage was about 9 hours. Holes were noticed in 21 of the 102 observed cells within the first hour, and in the next 15 cells during the second hour. During the first five, the bees pierced 89 cells. The opening of wax capping, from the first hole noticed to the complete uncovering of the freeze-killed prepupae, lasted from 20 minutes to 44 hours. The average was 10 hours and 30 minutes. Bees vigorously dragged a wax capping during this activity. Sometimes there were 2 or more bees on one cell at the same time. The boundary between the duration of removing dead brood from a cell and polishing the inside of this cell were difficult to recognize. The reason was that bees were rarely observed to move dead prepupae or their parts away from the cells and then to move them from the hive. Bees were seen to stay inside the cells for long periods of time. The cells were recognized as completely cleaned when bees were not focused on them and had stopped checking the insides. Some of the cells were empty. Both, the removing of dead brood and the polishing of the cells, lasted from 50 minutes up to 60 hours. Cell No. 3 was recognized as empty after 1 hour and 40 minutes of polishing. The highest number of cleaned cells was noticed between the 18 th and the 36 th hour after introduction of killed brood. The longest cleaning process of 88 hours occurred in cell No. 10. The queen was observed to lay eggs in 36 cells. The first egg laid was noticed 32 hours after the beginning of the cleaning process. The latest egg was laid in the 83 rd hour after introduction of killed brood. Nevertheless, queen did not lay an egg immediately after the cell cleaning T a b l e 1 Duration of successive stages and time of cell cleaning with dead brood (h:min) Stages of the cell cleaning process Time before clearing n Time the earliest -the latest Duration of a stage min - max mean SD 0:0-14:40 0:5-14:40 2:55 2:39 Recognition 0:5-14:40 0:18-24: 20 8:57 5:30 Uncapping 102 1:20-36:35 0:20-44:25 10:37 9:09 Cleaning 1:40-59:20 0:50-61:50 35:59 11:55 Egg laying by honeybee queen 36 32:41-82:27 2:35-67:30 17:10 12:42 n - number of observed cells Time - time from beginning of observation to start a particular stage of cleaning for the first (the earliest) and the last (the latest) of observed cells SD - standard deviation (h:min)

Journal of Apicultural Science 9 process was completed. The shortest time was 2 hours and 35 minutes in cell No. 39, and the longest after 67 hours in cell No. 58, about 17 hours on average (Tab. 1, Fig. 1). Some of the cells were being cleaned continuously during the cleaning process. However, this process was interrupted in most of the cells for several hours and then continued again; likewise for the successive stages of cleaning. Some of cells were ignored by bees during the process of cleaning. Bees had been cleaning the cells with different intensity. This resulted in a different length of the cleaning process in a different cells. DISCUSSION AND CONCLUSIONS Bees start cleaning cells when a dead brood appears in their colony, regardless of the reason of its death. The duration of cleaning process is different for every single cell. The reason is that bees frequently interrupt cleaning to perform other activities (Arathi and Spivak, 2001). The experimental bees also performed regular foraging flights. Robinson (1987) conducted similar research; but his work was limited only to monitoring the period of cleaning the cell after emergence of young worker bees, to the moment of egg lying by the queen. He divided this period into two stages: removing cocoons and polishing inside the cell. He stated that the average time for cleaning the cells was 41 minutes. In our work, the whole cleaning process of dead brood, took from 3 hours to almost 90 hours; the average was 46 hours. However, polishing only the inside of the cell, and preparing it for egg laying, took from 50 minutes to 62 hours (36 hours on average). It is important to note that a small bee colony was used in the present research what could have affected such differences in duration of cleaning process. In our research, the start of dead brood removal from the cells was seldom noticed. Borsuk et al. (2007) showed a short recording of bee hygienic behaviour and observed a bee worker carrying a piece of dead brood. As pointed Milne (1983), the mechanism of removal of the brood killed with different methods is not well known. He supposed that brood infected with Paenibacillus larva is probably eaten by bees and chalkbrood mummies are drawn out of the cells and then removed from the hive. Milne also observed that freeze killed brood is removed with both of the methods, but first of all it is eaten by bees. In our research, eating was probably the main method of clearing out dead brood, since bees were rarely noticed carrying out any fragments of prepupae. The phenomenon of unwanted brood removal through eating it, was first shown by Woyke (1963). He stated that bees eat diploid drone larvae. According to Woyke (personal information), soft brood of larvae, prepupae and soft parts of pupae are eaten, while hard parts of pupae are removed from brood cells. In our research the queen was frequently seen on the piece of comb with dead brood. She laid 6 first eggs between 32 nd and 35 th hour of observation while 56 cells were cleaned and probably prepared for egg laying. Subsequently, she was seen there between 39 th and 42 nd hour of observation. Then the queen laid 10 eggs while 64 cells were empty. Also in an experiment conducted by Woyke (1980) the queen laid new eggs in the empty cells in different periods of time after the diploid larvae were eaten. It is unknown what stimulates the queen to lay an egg in a particular cell. Woyke (1981) supposed that egg laying depended on weather and nectar conditions as well as origin of the queen.

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Journal of Apicultural Science 11 Woodrow A.H. (1941) - Behavior of honeybees toward brood infected with American foulbrood. Am. Bee J. 81: 363-366. Woyke J. (1963) - What happens to diploid drone larvae in a honeybee colony. J. apic. Res. 2(2): 73-76. Woyke J. (1980) - Effect of sex allele homo-heterozygosity on honeybee colony populations and on their honey production. 1. Favourable development conditions and unrestricted queens. J. apic. Res. 19(1): 51-63. Woyke J. (1981) - Effect of sex allele homo-heterozygosity on honeybee colony populations and on their honey production. 2. Unfavourable development conditions and restricted queens. J. apic. Res. 20(3): 148-155. PRZEBIEG OCZYSZCZANIA KOMÓREK PLASTRA Z MARTWEGO CZERWIU 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. S t r e s z c z e n i e Zachowanie higieniczne pszczół polegające na rozpoznawaniu, odkrywaniu i usuwaniu chorego lub martwego czerwiu z zakrytych komórek jest mechanizmem hamującym rozprzestrzenianie się choroby lub pasożyta w rodzinie pszczelej. W 2006 roku obserwowano przebieg usuwania czerwiu zabitego przez mrożenie, z komórek plastra umieszczonego w jedno-ramkowym ulu obserwacyjnym ze szklanymi ścianami. Szczegółowo obserwowano część plastra z 102 komórkami z zasklepionymi martwymi przedpoczwarkami. W okresie oczyszczania wyodrębniono etapy: rozpoznanie i zainteresowanie komórką, zgryzanie zasklepu żuwaczkami, usuwanie martwej przedpoczwarki i polerowanie wnętrza komórki. W niektórych z wyczyszczonych komórek obserwowano złożenie przez matkę jaja. Stwierdzono różne okresy rozpoczynania i kończenia kolejnych etapów oczyszczania poszczególnych komórek. Pszczoły całkowicie oczyściły komórki w czasie 3 do 85 godzin, a średnio w ciągu 46 godzin. Słowa kluczowe: zachowanie higieniczne, czerw mrożony, czas i etapy oczyszczania.