Praca oryginalna Original paper
The defensive behaviour of Apis mellifera L. wor-kers is a psychic trait that has been devoted a great deal of attention by researchers (12, 13). The high aggressiveness displayed by a colony that is being upset by an intruder multiplies its chances of survival. The research of worker defensive behavior also consti-tutes the basis for the selection by bee breeders aimed at obtaining calm colonies (11). On the other hand, workers do not produce offspring but the queens do. Reproductive conflicts between virgin queens are one of the main tools of natural selection (2, 14). Among bees, the conflict assumes the form of predominantly fatal fights (30). The elimination of excess queens reared in a colony involves: the departure of a queen with a swarm, the stinging of a queen by its rival in the queen cell or as a result of a mortal duel with the rival (10). Queens also fight in swarms containing more than
one queen (26). Aggressive behavior is observed between A. mellifera capensis egg laying workers and queens (18).
As we shown above honeybees show aggression during nest defense and during reproductive conflicts. Natural selection should favor both the efficacy of worker defensive response and the efficacy of queens in winning mutual fights. The question is whether the aggression displayed during nest defense has the same background as the aggression shown during reproduc-tive conflicts?
Hunt (12) enumerates 128 identified QTL candidate genes, located in three QTL regions (Sting 1, Sting 2 and Sting 3), that determine worker aggressiveness/ calmness. The genes located in the Sting1 region have the strongest affinity to stinging, affecting the develop-ment of the nervous system and the neurotransmitter secretion level. Thus, the Sting 1 region would not only determine the aggressiveness of workers but also that of queens.
Comparison of the defensive behaviour
of Apis mellifera L. workers and the aggression
of their queen sisters*
)
KORNEL KASPEREK, JERZY PALEOLOG, KRZYSZTOF OLSZEWSKI, GRZEGORZ BORSUK, ANETA STRACHECKA
The Life Science University in Lublin, ul. Akademicka 13, 20-280 Lublin
Kasperek K., Paleolog J., Olszewski K., Borsuk G., Strachecka A.
Comparison of the defensive behaviour of Apis mellifera L. workers and the aggression of their queen sisters
Summary
There are two situations in which bees fight and show aggression: protection of the nest and reproductive competition. This inspired the authors to verify the hypothesis that the intensity of workers defensive behavior corresponds with the aggressiveness of their queen sisters. Two calm Buckfast colonies (C1 and C2) and two aggressive European black bee hybrids colonies (A1 and A2) were used. The defensive behavior of workers was assessed with 45 sting tests for each colony. The aggressiveness of queens sisters of the workers from C1, C2, A1, A2 was assessed by observing queen pairs fighting in prepared arenas for 15 hours in the following combinations: C1C1, C2C2, A1A1, A2A2. Queen stings were clipped. In total, 176 pairs of queens were tested. Behaviors of the workers and their queen sisters were compared. The classification of the workers (several traits were considered), starting with the calmest and ending with the most aggressive (C1 < C2 < A1 < A2) differed from the aggressiveness classification of their queen sisters (A2 < C1 < C2 < A1). Consequently, the intensity of workers defensive behavior does not clearly correspond with the aggressive-ness of their queen sisters. This may be due to different physiological conditions and different emotive reactions developed in the course of evolution.
Keywords: Apis mellifera, defensive behavior, aggressive behavior, bee queens
*) A scientific study financed with the research fund for 2010-2011,
The mutual aggression of queens is unleashed by pheromones secreted from tergal glands located between the 3rd, 4th and 5th abdominal tergum (25). The
course and result of the duel are conditioned by: the age, body weight, insemination method and genetic origin of the queens, as well as by the age of the larvae from which the queens had emerged (9, 23, 29, 30). This is a proof of the complex nature of individual queen-selection mechanisms, as opposed to group mechanisms in the case of worker defense. Hence, the aggressiveness of queens and workers may be determined by different genes/mechanisms.
All findings mentioned above have caused that the authors decided to investigate the hypothesis that aggressiveness in workers corresponds with the aggres-siveness of their queen sisters (coefficient of relation-ship: R = 0.750.25) (31).
Material and methods
The study was carried out in 2007, 2008 and 2009 at the University of Life Sciences in Lublin (N: 51°13; E: 022°38). Two Buckfast bee (21) colonies were used (C1 and C2) that had been classified as very calm in the pre-liminary tests, in addition to two colonies of hybrids of unknown origin that had been classified as very aggressive (A1 and A2). The traits and behavior of the hybrids sug-gested a considerable genetic influence of the European black bee (A. mellifera mellifera; 27, 33). Throughout the experiment, the same queens headed the colonies. Each year, the population of each of the four colonies was reduced to the level of the least numerous one (7 fully populated combs) in order to offset the influence of the number of the bees on the results.
The first part of the study involved evaluation of the aggressiveness of the C1 and C2 and A1 and A2 workers by means of field sting tests. The experimental procedure was repeated 10, 15 and 20 times for each of the four colo-nies in 2007, 2008 and 2009, respectively. The second part involved the monitoring of duels of the queens that were the sisters of the workers analyzed in the first part. Labo-ratory cage tests were conducted on 176 pairs of queens, including 48 C1C1 pairs, 48 C2C2 pairs, 38 A1A1 pairs and 42 A2A2 pairs. Each pair was observed separately.
The field sting tests (part 1) were accomplished in the afternoon following the last nectar flow (approx. July, 15th).
The bees were stimulated with a triple hammer knock on the hive entrance. A sheepskin glove with a surface of approximately 500 cm2 was placed in front of the entrance.
A separate glove was used for each colony. After each test the glove was rinsed to remove the pheromone scent. The glove was moved in front of the entrance for two minutes, starting from the moment when the first sting was driven into it. Subsequently, the observer withdrew from the hive to assess the pursuit distance negotiated by the bees. The bees that attacked the glove were additionally filmed (Sony DCR-VX2100E). Freeze-frame images taken 30, 60, 90 and 120 seconds from the moment when the first sting perfo-rated the glove were used to determine the number of
attacking workers. This procedure was applied, since not all attacking bees leave their stings in the target. Some of them only strike the target trying to use their mandibles. The following characteristics were identified: (1) the time from the moment of stimulation until the first stinging [sec.]; (2) the number of stings left in the glove during two minutes from the moment of the first perforation [it.]; (3) the distance the bees negotiated in pursuit of the experi-menter [m]; (4) the number of bees directly attacking the glove 30, 60, 90 and 120 seconds following the first sting. In order to obtain queens for the laboratory cage tests (part 2), the larvae removed from the C1 and C2 along with the A1 and A2 colonies were introduced into queen-less nurse colonies. Two days following emergence, the queens were weighed (0.1 mg). The tips of their stings were clip-ped (CO2 anesthesia) to prevent fatal stinging during the further fights. The test started after the queens turned 7 days of age. Each 100 × 100 × 35 mm cage was divided into two arenas whose surfaces were covered with a wax foundation. Each arena was provided with a feeder with honey and water (1 : 1). Two queens marked with a felt-tip pen were let inside an arena. When creating pairs (C1C1, C2C2, A1A1 and A2A2), opponents with similar body weights were selected to minimize body weight influence on the fight result (30). Previous studies (22, 24) had confirmed that a fight of bee queens consists of alternate rounds and breaks. Therefore, the present study analyzed the following characteristics: (1) time from the introduc-tion of a queen pair into the arena until the start of the first charge [min.]; (2) mean duration of a single round (tR) [min.]; (3) mean duration of a single break (tB) [min.]; (4) total duration of all the rounds (ÓtR) [min.]; (5) total duration of all the breaks (Ót B) [min.]; (6) duration of a fight (tF; tF = ÓtR + Ót B) [min.]; (7) number of rounds during a fight.
The results were statistically verified using one-way ANOVAs combined with Duncans multiple range tests (28). The Least Significant Difference value (LSD) was estimated with Fisher LSD test (P £ 0.05).
It is difficult to compare the defensive behavior of entire colonies (workers) with the aggressiveness of particular queens originating from the same colonies (sisters of the workers). This is because the aggression of the workers (sting test) is a group trait, whereas the aggression of queens is an individual trait. Therefore, for a comprehensive comparison C1, C2, A1 and A2 workers and queens, the values of each characteristics were expressed as points in the following way. Mean values of each of the characteri-stics analyzed in the sting test were calculated for each of the four colonies separately (all repetitions during the three years). Additionally, the LSDs were computed for all these data sets. The mean values were then put in order ranging from those indicating the calmest behavior to those showing the most aggressive colony response. The calmest colony was awarded 0 points. If the subsequent mean differed (P < 0.5) by one LSD unit, the colony was awarded 1 point; if the difference was one and a half LSD units, the colony received 1.5 points, etc. The value of each successive mean was expressed as points in the same manner. The mean
values of each of the characteristics for the four types of queen pairs separately (C1C1, C2C2, A1A1 and A2A2 pairs) were arranged starting with the calmest indication up to the value denoting the highest aggressiveness. Points were awarded in the same way as in the case of the worker sting test.
Results and discussion
The A1 and A2 workers turned out to be more ag-gressive as regards to all the characteristics analyzed in the sting test. The colonies were ranked from the calmest to the most aggressive one (based on the point scale) as follows: C1 < C2 < A1 < A2 (Tab. 1). Workers need to be effectively stimulated to pass from a state of alarm and search to attacking (6). The speed of the reaction depends on individual response thre-shold and intensity of the stimulus (32). The C1 and C2 workers started attacking later and left fewer stings in the glove. This may be accounted for with either a lower intensity of the alarm stimulus, i.e. a lower volume of the pheromones secreted by the guard-bees (3, 12, 32) or with a high worker response threshold. Possibly a combination of both factors is involved. This is confirmed with lower numbers of the C1 and C2 bees attacking the glove and with shorter pursuit distances of the C1 and C2 workers as compared with the A1 and A2 workers.
Very modest differences were observed between the calm C1 and C2 colonies which had a high response threshold level. On the other hand, the differences were greater in the case of the aggressive colonies (lower response threshold level). The A2 colony recruited more bees for defense, remained in a state of stimula-tion longer and went farther in pursuit of the
experi-menter than the A1 colony. On the other hand, though it recruited fewer bees for the attack, the A1 colony inserted more stings in the target, as a majority of the bees ended the attack with a perforation (Tab. 1). The defensive behavior of bees usually culminates in an attack (13) that, apart from stinging, involves such reaction as: pursuit, mandible pinching, or running over the intruders body (4, 6). Additionally, the author observed venom sprinkling by A2. All in all, the A2 workers more often took advantage of intimidation, thus minimizing losses among bees that die following sting insertion. Similar observations were made by Cunard and Breed (7), who found that most Central European bees do not sting the intruder but keep harassing the enemy to successfully intimidate it. The comparison of the methods of attacking the target by the A1 and A2 workers confirmed that the number of stings left in the target is not a sufficient measure of the defensive response, since one of the strategies of nest defense may be intimidation.
The ranking starting with the calmest and ending with the most aggressive queens (based on the point assessment) was as follows: A2A2 < C1C1 < C2C2 < A1A1 (Tab. 2), i.e. inversely to the case of the workers (C1 < C2 < A1 < A2; Tab. 1). Interestingly, the A2A2 queens were the calmest, while the A2 workers were the most aggressive. The differences between the C1C1, C2C2 and A1A1 queens were, in turn, inconsiderable. Measured with the time until the beginning of the first attack, the stimulus response speed of the queens from the aggressive colonies did not significantly differ from the speed of reaction of the queens from the calm colonies (Tab. 2). Hence, the stimulus response threshold was similar in the
Tab. 1. Aggressiveness of the worker bees (entire colonies) and its point assessment
s c it s ir e t c a r a h C ) s n a e m e h t f o h c a e r o f 5 4 = n ( y n o l o C 1 C C2 A1 A2 E S ± n a e m points mean±SE points mean±SE points mean±SE points e h t li t n u n o it a l u m it s f o t n e m o m e h t m o rf e m i T ]. c e s [ e v o l g e h t o t n i g n it s t s ri f e h t f o n o it r e s n i 22.6a±3.0 0.16 24.2a±4.07 0 10.5b±2.5 1.58 11.87b±2.64 1.40 o w t g n ir u d e v o l g e h t n i tf e l s g n it s f o r e b m u N n o it a r o fr e p t s ri f e h t f o t n e m o m e h t m o rf s e t u n i m 7.0b±1.1 0.15 6.5b±1.0 0 15.6a±1.58 2.84 15.4a±1.0 2.77 f o ti u s r u p n i d e t a it o g e n s e e b e h t e c n a t s i d e h T ] m [ r e t n e m ir e p x e e h t 13.4c±1.5 0 15.9c±1.7 0.20 59.5b±4.0 3.76 125.9a±7.5 9.18 r e tf a s e t u n i m 5 . 0 e v o l g e h t n o s e e b f o r e b m u N g n i g n it s t s ri f e h t 9.8b±1.1 0 14.4b±1.9 0.41 17.6b±1.8 0.71 50.4a±7.4 3.67 e h t r e tf a e t u n i m 1 e v o l g e h t n o s e e b f o r e b m u N g n i g n it s t s ri f 12.2c±1.1 0 16.6c±1.9 0.23 37.0b±4.3 1.34 107.8a±12.3 5.17 r e tf a s e t u n i m 5 . 1 e v o l g e h t n o s e e b f o r e b m u N g n i g n it s t s ri f e h t 15.5c±1.6 0 21.3c±2.6 0.20 66.3b±8.5 1.76 147.4a±18.6 4.57 e h t r e tf a s e t u n i m 2 e v o l g e h t n o s e e b f o r e b m u N g n i g n it s t s ri f 19.7c±2.3 0 24.2c±3.2 0.12 96.2b±17.6 1.99 169.1a±20.8 3.89 S T N I O P L A T O T 0.31 1.16 13.98 30.65
Explanations: A1 and A2 colonies of aggressive hybrids; C1 and C2 calm Buckfast colonies; a, b, c the differences are significant in rows at P £ 0.05 (Duncan test). The highest point score corresponds with the greatest aggressiveness
A1A1, A2A2, C1C1 and C2C2 queens but different in the A1, A2, C1 and C2 workers. The C1C1, C2C2 and A2A2 queens remained stimulated for a shorter period of time than the A1A1 queens, which fought for a longer time, with longer rounds of their fights and, consequently, a longer total duration of the clash (Tab. 2). The A1A1 queens may have had a slightly higher level of aggression, manifested, however, only through remaining stimulated for a longer time.
Summing up, the intensity of the defensive behavior of the workers representing the particular colonies did not coincide with the aggressiveness of their queen sisters. We may venture the conclusion that different mechanisms with different genetic grounds control worker and queen aggression.
Alaux et al. (1) found the aggressiveness and aggression-related gene expression of workers to rise along with their age. Paleolog (23) observed the level of aggressiveness of queens to rise to the maximum at insemination and subsequently fall. Thus, aggressive-ness expression during the life-span of a queen and that of a worker is different. The aggression of both queens and workers has a strong emotional background (5, 15, 16) consolidated in evolution. Emotions and reactions to stimuli are developed in the process of sensitization related to learning (8, 24). Animal sur-vival depends on the capacity of the brain to adapt to environmental changes (17). For a queen bee, this occurs when it meets the other queen sisters, not during nest defense as is the case with the workers (30). For reproductive success, a queen must survive such a confrontation, whereas a worker often has to perish. Different pheromones release aggression in queens and workers (34). In small doses, queen phero-mones may also act as repellents to workers (19, 20); in larger quantities they may induce worker
aggres-s c it s ir e t c a r a h C s ri a p n e e u q f o s e p y T ) 8 4 = n ( 1 C 1 C C2C2(n=48) A1A1(n=38) A2A2(n=42) E S ± n a e m points mean±SE points mean±SE points mean±SE points n e e u q a f o n o it c u d o rt n i e h t m o rf e m i T e h t f o tr a t s e h t li t n u a n e r a e h t o t n i ri a p ] n i m [ e g r a h c t s ri f 70.0 a±11.2 0.28 52.8a±9.9 0.8 79.2a±16.1 0 74.7a±10.6 0.14 t h g if a g n ir u d s d n u o r f o r e b m u N 12.7b±1.1 0.07 16.9a±1.4 1.91 10.3b±0.8 0 12.1b±1.3 0.54 ]. n i m [ d n u o r e l g n i s a f o n o it a r u D 25.5b±2.8 1.55 15.1c±1.3 0 39.3a±3.4 3.6 22.2b±2.3 1.06 ]. n i m [ h s a l c e h t f o n o it a r u D 322.9ab±30.8 0.83 254.6b±23.1 0 402.3a±38.1 1.78 269.2b±26.2 0.18 ]. n i m [ k a e r b e l g n i s a f o n o it a r u D 24.9a±2.4 0.3 17.4b±1.3 1.54 25.64a±3.08 0.19 26.8a±2.3 0 ]. n i m [ d o ir e p g n it s e R 291.2a±30.4 0.08 275.5a±29.0 0.27 237.2a±25.5 0.73 298.1a±29.7 0 ]. n i m [ n o it a r u d t h g if e ri t n E 614.2ab±30.2 0.92 530.1b±27.3 0 639.5a±36.2 1.27 567.4ab±28.9 0.43 S T N I O P L A T O T 4.03 4.52 7.57 2.35
Tab. 2. Aggressiveness of the bee queens and its point assessment
Explanations: A1 and A2 queens originating from the two aggressive colonies of local bee hybrids; C1 and C2 queens originating from the two calm Buckfast colonies. C1C1, C2C2, A1A1, A2A2 combinations of tested queens (pairs); a, b, c the differences are significant in rows at P £ 0.05 (Duncan test). The highest point score corresponds with the greatest aggressiveness
sion (35). In the context of the above arguments, the results of the present paper show that physiological differences and different emotional reactions developed in evolution underlie queen and worker aggression.
References
1.Alaux C., Sinha S., Hasadsri L., Hunt G. J., Guzman-Novoa E., DeGrandi--Hoffman G., Uribe-Rubio J. L., Southey B. R., Rodriguez-Zas S., Robinson G. E.: Honey bee aggression supports a link between gene regulation and behavioral evolution. Proc. Natl. Acad. Sci. USA 2009, 106 (36), 15400--15405.
2.Archer J.: The behavioural biology of aggression. Cambridge Univ. Press. 1988.
3.Arechavaleta-Velasco M. E., Hunt G. J.: Genotypic variation in the expres-sion of guarding behavior and role of guards in the defensive response of honey bee colonies. Apidologie 2003, 34, 439-447.
4.Breed M. D., Guzman-Novoa E., Hunt G. J.: Defensive behavior of honey bees: organization, genetics, and comparison with other bees. Annu. Rev. Entomol. 2004, 49, 271-298.
5.Broom D. M., Johnson K. G.: Stress and animal welfare. Kluwer Academic Publishers. Netherlands 2000.
6.Collins A. M., Rinderer T. E., Tucker K. W., Sylvester H. A., Lackett J. J.: A model of honeybee defensive behavior. J. Apic. Res. 1980, 19 (4), 224--231.
7.Cunard S. J., Breed M. D.: Post-Stinging Behavior of Worker Honey Bees (Hymenoptera: Apidae). Ann. Entomol. Soc. Am. 1998, 91, 754-757. 8.Dietemann V., Zheng H. Q., Hepbourn C., Hepbourn H. R., Jin S. H.,
Crewee R. M., Radloff S. E., Fu L. H., Pirk Ch. W.: Self assessment in insects: Honeybee queens know their own strength. PLoS One Jan 2008, 9, 3(1): e1412, 1-5.[On-line] http://www.plosone.org/.
9.Gilley D. C.: The behavior of honey bees (Apis mellifera ligustica) during queen duels. Ethology 2001, 107, 601-622.
10.Gilley D. C., Tarpy D. R.: Three mechanisms of queen elimination in swarming honey bee colonies. Apidologie 2005, 36, 461-474.
11.Guzman-Novoa E., Page R. E.: Backcrossing Africanized honey bee queens to European drones reduces colony defensive behavior. Ann. Entomol. Soc. Am. 1993, 86, 352-355.
12.Hunt G. J.: Flight and fight: A comparitive view of the neurophysiology and genetics of honey bee defensive behavior. J. Insect. Physiol. 2007, 53, 399-410.
13.Kastberger G., Thenius R., Stabentheiner A., Hepburn R.: Aggressive and docile colony defense patterns in Apis mellifera. A retreater-releaser concept. J. Insect. Behav. 2009, 22, 65-85.
14.Krzanowska H., £omnicki A. (red.): Zarys mechanizmów ewolucji. PWN, Warszawa 2002.
15.Lipiñski Z.: Essence and mechanism of nest abandonment by honeybee swarms. Edit. by an author. Druk Blenam. Olsztyn, Poland 2001.
16.Lipiñski Z.: The emotional nature of the worker honeybee (Apis mellifera L.) J. Apic. Sci. 2006, 50 (1), 49-61.
17.Matsuzaki M.: Factors critical for the plasticity of dendritic spines and memory storage. Neurosci. Res. 2007, 57, 1-9.
18.Moritz R. F. A., Brewe R. F., Pflugfelder J.: Lethal fighting between honey-bee queens and parasitic workers (Apis mellifera). Naturwissenschaften 2003, 90, 378-381.
19.Moritz R. F. A., Crewe R. P., Hepburn H. R.: Attraction and repellence of workers by the honeybee queen (Apis mellifera L.). Ethology 2001, 107, 465-477.
20.Moritz R. F. A., Crewe R. P., Hepburn H. R.: Queen avoidance and mandibu-lar gland secretion of honeybee workers (Apis mellifera L). Insect Society 2002, 49, 86-91.
21.Österlund E.: Brother Adam and His Buckfast Bee. Am. Bee J. 1983, 123 (2), 85-88.
22.Paleolog J.: Behavioural characteristics of honey bee (Apis mellifera) colo-nies containing mix of workers of divergent behavioural traits. Animal Science Papers and Reports 2009b, 27 (3), 237-248.
23.Paleolog J.: Honeybee queen docility noticed during queen duels. J. Apic. Sci. 2009a, 53 (2), 97-104.
24.Paleolog J., Kasperek K., Lipinski Z.: The psychological dimension of duels between western honeybee queens with blunted and non-blunted stings. J. Apic. Sci. 2011, 55 (2), 85-95.
25.Pflugfelder J., Koeniger N.: Fight between virgin qeens (Apis mellifera) is initiated by contact to the dorsal abdominal surface. Apidologie 2003, 34, 249-256.
26.Rodinow W., Szabarszow I.: Moje pszczo³y. PWRiL, Warszawa 1989. 27.Ruttner F.: Biogeography and Taxonomy of Honeybees. Springer-Verlag.
Berlin 1988.
28.SAS Institute SAS/STAT Users Guide release 9.13. Cary, NC, Statistical Analysis System Institute, license 86636.
29.Tarpy D. R., Hatch S., Fletcher D. J. C.: The influence of queen age and quality during queen replacement in honeybee colonies. Anim. Behav. 2000, 59, 97-101.
30.Tarpy D. R., Mayer M. K.: The effects of size and reproductive quality on the outcomes of duels between honey bee queens (Apis mellifera L.). Ethol. Ecol. Evol. 2009, 21, 147-153.
31.Tautz J.: Fenomen pszczó³ miodnych. Galaktyka Sp. z o. o. Poznañ 2008, 251-259.
32.Wager B. R., Breed M. D.: Does honey bee sting alarm pheromone give orientation information to defensive bees? Ann. Entomol. Soc. Am. 2000, 93, 1329-1332.
33.Wilde J., Bratkowski J., Siuda M.: Defensive behavior of 3 breeds of Apis mellifera L. Annales UMCS Lublin. Sectio EE Zootechnica 21 (2), 65, 87-93.
34.Winston M.: The biology of the honey bee. Harvard University Press. Cambridge, Massachuesetts, London, England 1987.
35.Vaitkeviciene G., Budriene A.: Age-related changes in response to queen pheromone and in retrocerebral complex of Apis mellifera L. workers. Pheromones. 1999, 6, 39-46.
Corresponding author: Jerzy Paleolog, ul. Akademicka 13, 20-280 Lublin; e-mail: jerzy.paleolog@up.lublin.pl
