Journal of Apicultural Research 51(4): 359-361 (2012) © IBRA 2012 DOI 10.3896/IBRA.1.51.4.11 NOTES AND COMMENTS First evidence of hygienic behaviour in the dwarf honey bee Apis florea Jerzy Woyke1*, Jerzy Wilde2 and Mananya Phaincharoen3 1 Apiculture Division, University of Life Science, 166 Nowoursynowska, 02-787 Warsaw, Poland. Apiculture Division, University of Warmia and Mazury, 10-710 Olsztyn, Poland. 3 King Mongkut's University of Technology, Thonburi, Ratchaburi Campus, Bangkok 10140, Thailand. 2 Received 18 March 2012, accepted subject to revision 25 June 2012, accepted for publication 4 July 2012. *Corresponding author: Email: jerzy_woyke@sggw.pl Keywords: Apis florea, hygienic behaviour, freeze-killing, pin-killing, migratory honey bees Both of the (hive) cavity-nesting honey bees Apis mellifera and A. cerana display hygienic behaviour which is seen by the removal of dead brood parasitized by Varroa destructor or killed by bacterial diseases (Spivak, 1996; Evans and Spivak, 2010). To the contrary, Woyke (1996) and Woyke et al. (2004) found that the migratory open-air nesting honey bees A. dorsata and A. laboriosa do not open sealed cells containing dead brood killed by the parasite Tropilealaps clareae or by any other disease. It is believed that this behaviour prevents the spread of parasites and diseases inside the nest. The third migratory open-air nesting dwarf honey bee, A. florea is parasitized by the mite Euvaroa sinhai (Akratanakul and Burgett, 1976; Ahmadi, 1988) and is infected by some fungi (Alizadeh and Mossadegh, 1994). Woyke et al. (2011) suggested that A. florea removes dead brood, but no data were presented. In the present Fig. 1. Freeze-killed brood inserted in the comb of Apis florea colony study we therefore investigated the hygienic behaviour of the dwarf No. 3, 9 March 2011, Ratchaburi, Thailand. bee A. florea. The investigation was carried out in Thailand, at the Ratchaburi campus of King Mongkut’s University of Technology in Thonburi different time intervals. Established colonies had clearly removed higher percentage of killed brood in 24 hours than the transferred (13º 59' N, 99º 51' E), from 9 to 13 March 2011. Eight A. florea colonies ones. In particular, the established colonies had less than 5% of were investigated. Three colonies were naturally established on the freeze-killed sealed brood left 24 h after the brood was reinserted into campus while five colonies were transferred from an area 1.5 km the combs. away. The investigation of the transferred colonies started 5 days after they were introduced on campus. Comb pieces of sealed brood A one-way ANOVA of the results of all 8 colonies, showed that 24 h after killing the brood, the method of killing (frozen, or killed by pins were deep freeze-killed (Fig. 1) or pin-killed using pins with 0.2 mm or of 0.7 or 0.2 mm) significantly affected the percentage of the remaining 0.7 mm in diameter. The removal of the killed brood was checked in sealed brood (F5, 23 = 13.30, P = 0.000). Significant differences were time intervals between 24 and 60 h after it had been presented to the also detected between the established and the transferred colonies. In bees. One-way ANOVA was performed on the data concerning the 24 particular, 5.5, 5.3, and 6.2 times significantly more brood, killed by hours interval and the two-way ANOVA on data concerning results of the three methods, was left in the transferred colonies than in the pins of two diameters in three intervals. For statistical calculations, the established ones. The Duncan’s multiple range tests, however, did not percentages of the remaining killed brood were transformed according show significant differences between the means of the remaining to the arcsine function. Correlation coefficients were also calculated. Table 1 summarizes all data concerning percentages of sealed brood killed by the three methods which remained in the cells after brood killed by the three methods in the established colonies. No significant difference was also detected between percentages of left freeze-killed brood or pin-killed brood with the thick pin of 0.7 mm 360 Woyke, Wilde, Phaincharoen Table 1. Remains of sealed brood killed by three methods in A. florea colonies. Different capitals (A, B, C) indicate significant differences (p < 0.05) between means in the same column, concerning the method of killing the brood. Different small letters (a, b, c) indicate significant differences (p < 0.05) between means in the same row, concerning the time of checking the brood. Method No No of killing combs cells Frozen E 3 376 Pin 0.7 E 3 150 Pin 0.2 E 3 150 6.7Ab Frozen T 5 807 19.4Ba - 0.0 - Pin 0.7 T 5 250 21.2Bb 10.2Ba 4.0Aa 0.0 Pin 0.2 T 5 250 41.4Cc 15.6Cb 5.6Aa 0.0 Mean % sealed cells left after hours 24 42 48 60 3.5Aa - 0.0 - 4.0Ab 0.7Aa 0.0 0.0 2.0Aa 0.0 0.0 Established colonies Transferred colonies diameter within the transferred colonies, but twice as much brood been left. No significant interaction was found between the two factors remained after being pierced with the thin, 0.2 mm pin (Table 1). (F2, 29 = 2.25, p = 0.127). In the established colonies, 98.0-99.3% of Thus, the bees from the transferred colonies removed brood pinned pin-killed brood was removed within 42 h and all killed brood was with the thin pin much less effectively than pinned with the thick pin removed within 48 h. In the transferred colonies 94.4-96.0% of or freeze-killed. The same effect of the pin difference in diameter is pin-killed brood was removed within 48 h and all killed brood was shown also later, after 42 h (F3, 15 = 15.10, p = 0.000). Forty-eight removed within 60 h (Table 1). The correlation between the percentages hours after killing, no killed brood remained in the established of brood left in the 5 transferred colonies, after being pierced with the colonies, while a very small proportion of pin-killed brood (4.0-5.6%) thick or the thin pin (0.7 mm or 0.2 mm), was significant; r = 0.95, still remained in the transferred colonies. The removal time of the df = 4, p = 0.013. However, no significant correlation was found killed brood was affected by the method of killing. All frozen brood in between the percentage of the remaining freeze-killed brood and the the 8 colonies was removed within 48 h after being introduced into percentage of remaining brood pierced with either the thick pin (0.7 mm; the combs (Fig. 2). r = 0.16, df = 4, p = 0.800) or the thin pin (0.2 mm; r = 0.26, df = 4, p = 0.676). The slower removal of dead brood by the transferred colonies than by the established ones was unexpected. No proven explanation exists as to whether this was just variation or whether two populations of different hygienic behaviour were present, or perhaps that the transportation affected the results. Further investigations are necessary. In conclusion, all 8 colonies removed 89% of the freeze-killed brood within 24 h after it had been inserted into the combs and 95% of the pin-killed brood within 48 h. Thus, those A. florea colonies have to be considered as hygienic honey bees. Further investigations are desirable concerning the variation of hygienic behaviour of different populations of A. florea, as well as the behaviour in shorter intervals of 8 and 16 h, which could give clearer and more profound results. Fig. 2. Empty piece of comb 48 h after insertion of freeze-killed brood into colony No. 3. A two-way ANOVA on the data concerning results of pins of two Acknowledgments The research was supported by the Polish Ministry of Science and diameters in three intervals, showed that diameter of the pins Higher Education, research project No 528.0113.0811, and by the (F1, 29 = 9.43, p = 0.005) as well as the checking time (F2, 29 = 34.17, National Research University Project of Thailand’s Higher Education p = 0.000), significantly affected the percentage of brood which had Commission Office. Hygienic behaviour of Apis florea References 361 WOYKE, J (1996) Different reaction of Apis dorsata and Apis mellifera to brood infestation by parasitic mites. In Proceedings of 3rd Asian AHMADI, A A (1988) Parasites of Apis florea in Iran. Bee World 69: 159-161. 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