1 Annals of Warsaw University of Life Sciences SGGW Animal Science No 46, 2009: (Ann. Warsaw Univ. of Life Sc. SGGW, Anim. Sci. 46, 2009) The impact of the sequence of particular drone semen administration on number of their own progeny after instrumental insemination MACIEJ SIUDA*, JERZY WILDE*, BEATA B K*, MA GORZATA KOBYLI SKA** *Apiculture Division, Faculty of Animal Bioengineering, Warmia and Mazury University in Olsztyn **Department of Quantitative Methods, Faculty of Economic Sciences, Warmia and Mazury University in Olsztyn Abstract: The impact of the sequence of particular drone semen administration on number of their own progeny after instrumental insemination. During the mating flight, a queen copulates with several, sometimes more than ten, drones. It is known that a queen preserves semen from all the drones with which she had copulated. However, it is not clear if the particular drone position in the mating sequences influences the number of this male s progeny. Investigations were performed in the apiary of Apiculture Department of Warmia and Mazury University in Olsztyn (Poland) from 1 July till 30 Sept The experiment was carried out on 36 Carniolan queens (Apis mellifera carnica). Each of them was instrumentally inseminated with semen collected from nine drones. The drones represented two bee races Apis mellifera ligustica and A.m.carnica that have different body colouring. It was found that each of the drones which copulated with a queen had a chance for acquiring progeny. Although the findings indicated that the progeny of drones semen which was the last to be injected into queen s oviduct had the smallest range. Key words: drones, queens, insemination, bee workers, colour inheritance, number of progeny. INTRODUCTION Within a healthy bee colony drones are produced only during the reproduction period May to August. Their main function is to mate and inseminate the young queens with semen. Queen and drone copulations occur in the air during mating flights (Soczek 1958). A queen is successively mated with several drones (Woyke 1960) during nuptial flights. Except for the mating flight periods, drones do not display interest in young queens whether they are in nest or its vicinity (Zmarlicki and Morse 1963). Virgin queens fly to drone congregation areas to get mated with mature drones. Drones aggregate in the chosen areas which can be used even for several years (Ruttner 1966, Loper 1985). Drones mark congregation areas and flight tracks with a specific pheromone (Lensky et al. 1985). Such a mechanism probably facilitates the young queens to find the drone congregation area. Drones congregate around a flying queen lured by her secreted pheromones (Butler 1976). Koeniger et al. (1979) managed to film the copulation process of the honey bees. After copulation, the drone dies from a total tectonic contraction of abdominal muscles, that is necessary for copulation
2 110 M. Siuda et al. apparatus extrusion and semen injection into oviducts of queen (Woyke 1958). The biology of queens insemination makes extreme demands on the drone. To convey its genes, a drone must overcome the great concurrence of the other drones. Koeniger s et al. (1979) film showed that the drones are not waiting passively on their chance but they are fighting to get the best position all the time. In order to do this, they hit their heads against the competing drones, not only those nearby but even at the moment of with a queen (Koeniger et al. 1979). After her mating flight, the queen comes back to the hive and pushes the collected semen inside her body by contracting the oviduct s muscles. All the time a small amount of semen (ca. 6%) penetrates into spermatheca of queen (Woyke 1960). The queen preserves semen from all the drones with which she has mated (Jasi ski 1994). This happens as the semen from each drone is mixed in queen oviducts as a consequence of injection force and contractions of oviduct s muscles (Jasi ski 1994, Woyke 2008b). In spite of this mixing process, the quantity of semen that enters into queen s spermatheca from particular drones may be differentiated. So, this raises the question if a place of a particular drone in mating sequence influences quantity of its progeny. The aim of this study was to determine the possible impact of the order of a particular drone s semen injection during instrumental insemination on the amount of its own progeny. MATERIAL AND METHODS The study was carried out in apiary of Apiculture Department of Warmia and Mazury University in Olsztyn between 1 July and 30 September Thirty six Carniolan queens (Apis mellifera carnica) were used. The queens were instrumentally inseminated with semen from nine successive drones according to the following schedule: Group I (12 queens) the first drone of Italian race (A.m. ligustica) and next eight Carniolan drones (A.m.carnica); Group II (12 queens) first four Carniolan drones followed by one Italian drone and again four Carniolan drones; Group III (12 queens) first eight Carniolan drones followed by one Italian drone. During instrumental insemination, semen collected from one drone was singly injected into queen s oviduct and the insemination needle was later thoroughly rinsed with physiological salt solution. After insemination queens were introduced into a new settled nucleus. Combs of sealed brood with the progeny of investigated queens were isolated in an incubator before the young bees emergence. Emerging worker bees were counted and recorded as light (after Italian drone) and dark (after Carniolan drones) coloured separately. In order to verify the hypothesis that a queen s insemination by a given sequence of drones does not influence percentage of light and dark coloured workers, a chi- -quare independence test was used. Statistical calculation was performed for three independent trials. RESULTS The average distribution of light-coloured bees in Groups I and II were similar to each other and amounted to 28.4
3 The impact of the sequence of particular drone semen administration and 27.9 individuals that was respectively 22% and 23% of the emerging workers population. In comparison with groups I and II, light-coloured worker population distribution in the third group was lower with an average of 19.7 individuals. It constituted 20% of all worker population emerging in that group (Tab. 1). A statistical (chi-square) independence test for 3 independent trials to verify hypotheses showed that the sequence of semen injections during instrumental insemination did not influence a percentage of lightand dark-coloured workers. The Chi- -square test value amounted Critical values for (2-1)(3-1) = 2 freedom degree and reliance range p = 0.05 and p = 0.01 are and respectively. The performed test showed no influence on light- and dark-coloured workers percentage in their progeny by semen injection sequence during instrumental insemination of queens. A wide span has been found between extreme results within the progeny of particular queens. The greatest span appeared in the second group which amounted 41.1 (Fig. 1) having both the least (8%) and the greatest (49.1)% contribution of Light - coloured bees Dark - coloured bees FIGURE 1. Mean value as well as minimal and maximum values of light and dark-coloured bees proportions (%) Explanations: 1 the first drone of Italian race (A.m. ligustica) and next eight Carniolan drones (A.m. carnica), 2 first four Carniolan drones followed by one Italian drone and again four Carniolan drones, 3 first eight Carniolan drones followed by one Italian drone TABLE 1. Total and average number and percentage of the light and the dark-coloured bees Order of semen n Light-coloured bees Dark-coloured bees injection individuals % individuals % 1I+8C n = 341 n=1153 4C+1I+4C n = n= C+1I n = n = 942 Explanations: 1I + 8C the first drone (of the light colour) of Italian race (A.m. ligustica) and next eight Carniolan drones (A.m. carnica), 4C + 1I + 4C first four Carniolan drones followed by one Italian drone and again four Carniolan drones, 8C + 1I first eight Carniolan drones followed by one Italian drone. 80
4 112 M. Siuda et al. light-coloured bees in progeny of particular queens. The least differentiated bees were found in the third group where the extreme results values differed by Within this group, the least proportion of light-coloured bees amounted by 10.7% and the highest by 30.9% of particular queen progeny. DISCUSSION Drones were often not appreciated due to their haploid nature and their lack of direct engagement in bee colony productivity. Only in recent years has more interest has been shown for the drones presence for intensive apiculture production (Woyke 2008a). Drones, originating from unfertilized eggs, were neglected in bee genetics they in favour of the genetics of their mothers (Rinderer 1986, Paleolog 1996 a and b). Bees body colour depends on one pair of the main genes and seven modulator genes (Woyke 1977). As a result of their interaction ten classes of bee colouring exists with various proportions of yellow and black colours. The Carniolan bees used in our studies were generally dark-coloured although they can possess some more or less yellowish tones. In this study, all bees which did not have an entirely dark body colour were classified as the progeny of Italian drones which characterize themselves with light, yellow body colour. That is why a theoretical 11% proportion of light-coloured workers was found only within progeny of two queens. Jasi ski (1994) found the same proportion of mutant worker bees after inseminating queens with mutant drones semen administered in various sequences. In this experiment considerable seasonal variation was observed in the number of bees of dark and yellow colour. CONCLUSIONS Our study has shown that every drone whose semen was inseminated to a queen has a chance to produce own progeny. The findings indicated that the progeny of drones semen which was injected into queen s oviduct as the latest, had the smallest range. REFERENCES BUTLER C.G The world of the honeybee. Collins. London, ss JASI SKI Z. 1994: Opró nianie jajowodów przez matki naturalnie i sztucznie unasieniane. Biuletyn Sztuczne Unasienianie Matek Pszczelich. 1: KOENIGER G., KOENIGER N., FABRITIUS M. 1979: Some detailed observations of mating in the honeybee. Bee Word, 60: LENSKY Y., CASSIER P., NOTKIN M., DELORME-JOULIE C., LEVINSOHN M., 1985: Pheromonal activity and fine structure of the mandibular glands of honeybee drones (Apis mellifera L.) (Insecta, Hymenoptera, Apidae). J. Insect Physiol., 31: LOPER G.M. 1985: Influence of age on the fluctuation of iron in the oenocytes of the honey bee (Apis mellifera) es. Apidologie, 16: PALEOLOG J. 1996a: Specyfika pracy hodowlanej w pszczelnictwie. I. Ocena warto ci u ytkowej i hodowlanej. Przegl. Hod., 64(4): PALEOLOG J. 1996b: Specyfika pracy hodowlanej w pszczelnictwie. II. Selekcja, dobór do kojarze. Przegl. Hod., 64(5): RINDERER T.E. 1986: Bee genetics and breeding. Acad. Press., Orlando (Floryda): RUTTNER F. 1966: The life and flight activity of drones. Bee Word, 47: SOCZEK Z. 1958: Wp yw niektórych czynników na loty i kopulacj matek pszczelich. Pszczeln. Zesz. Nauk., 2(3): WOYKE J. 1958: Przebieg kopulacji u pszczó. Pszczeln. Zesz. Nauk. 2(1): 142.
5 The impact of the sequence of particular drone semen administration WOYKE J. 1960: Naturalne i sztuczne unasienianie matek pszczelich. Pszczeln. Zesz. Nauk 4(3-4): WOYKE J. 1977: The heredity of colour patterns in the honey bee. Itern. Symposium on Genetics, selection and reproduction of the honey bee. Moscow 1976: 4955, Apimondia Publ. House, Bucharest. WOYKE J. 2008a: Genetyka pszczó. [w] Hodowla pszczó. Praca zbiorowa pod redakcj Wilde i Prabucki. PWRi L. Pozna : WOYKE J. 2008b: Why the eversion of the endophallus of drone stops at the partly everted stage and significans of this. Apidologie, 39(6): ZMARLICKI C., MORSE R.A. 1963: Drone congregation areas. J. Apic. Res., 2: Streszczenie: Wp yw kolejno ci wprowadzania nasienia trutni podczas sztucznego unasieniania na ilo potomstwa w asnego. Matka pszczela podczas lotu godowego kopuluje z kilkoma lub nawet kilkunastoma trutniami. Wiadomo, e matka pszczela przechowuje nasienie od wszystkich trutni z którymi kopulowa a, natomiast nie jest jasne czy kolejno kopuluj cych trutni ma wp yw na ilo powsta ego po nich potomstwa. Celem pracy by o okre lenie wp ywu kolejno- ci wprowadzania nasienia od poszczególnych trutni podczas sztucznego unasieniania na ilo ich potomstwa. Do wiadczenie wykonano w pasiece Katedry Pszczelnictwa UWM w Olsztynie w okresie od do roku. W do wiadczeniu u yto 36 matek rasy krai skiej (Apis mellifera carnica). Matki te unasieniono sztucznie nasieniem pobieranym od 9 trutni dwóch ras Apis mellifera ligustica i A. m. carnica ró ni cych si ubarwieniem. Stwierdzono, e ka dy trute unasieniaj cy matk ma szans na pozostawienie potomstwa. Potomstwo po trutniach, których nasienie wprowadzane jest do jajowodów matki jako ostatnie maj, charakteryzowa o si najmniejszym rozst pem. MS. received November 12, 2009 Authors address: Uniwersytet Warmi sko-mazurski ul. S oneczna 48, Olsztyn Poland