Received 25 March 2010; Accepted 19 October 2011

Journal of Apicultural Science 5 MICROSCOPIC IMAGE OF HONEYBEE DRONE SPERMATOZOA IN THREE DILUENTS G r z e g o r z B o r s u k 1, K r z y s z t o f O l s z e s k i 1, A n e t a S t r a c h e c k a 1, J e r z y P a l e o l o g 1, M a ri u s z G a g o 2, 3 1 Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, 2 Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland 3 Department of Cell Biology, Institute of Biology and Biotechnology, Maria Curie Sk odowska University, 20-033 Lublin, Poland e-mail: grzegorz.borsuk@up.lublin.pl INTRODUCTION Communities of social insects are characterised by a polyandric mating system (Strassmann, 2001). In natural mating, a queen bee may copulate with many drones (Ruttner, 1969; Moritz, et al., 1996; Schl ns, et al., 2004). This is related to polyandry (Triasko, 1951; Borsuk et al., 2011a,b), which is most highly organized in bees (Fuchs and Moritz, 1999; Koeniger and Koeniger, 2000; Palmer and Oldroyd, 2000). Natural mating occurs in the air, which means a person cannot control the Received 25 March 2010; Accepted 19 October 2011 S u m m a r y Microscopic analysis of the behaviour of spermatozoa diluted in diluents was conducted in vitro and in vivo. Additionally, an attempt was made to determine the factors that may affect semen coagulation in the instrumental insemination process. Under a microscope, the following semen smear groups were observed: 1. according to drone age; 2. in commercial diluents: saline, SAFE boar semen diluents; 3. 0.2 l semen with spermathecal uid from 4-6 virgin queen bees; 4. from partially lled spermatheca - we used spermatozoa after reinsemination. Twelve smears in each group were viewed under the OLYMPUS microscope. The microscope was coupled with a digital camera for lming and taking photographs of the semen. Motility of spermatozoa/semen was observed in the preparations. A stopwatch was used to measure the length of time between preparations and the beginning of sperm movement until full cessation of movement. Semen from young drones did not coagulate. Semen coagulation most frequently occurred (44%) when mixing the semen from old and mature drones. After 12 min, cessation of spermatozoa movement was observed in the semen diluted with saline. Spermatozoa in the boar semen diluents exhibited motility for 29 min. After reinsemination, spermatozoa displayed circular arrangement. Key ords: semen smears, semen coagulation, reinsemination, drone, Apis mellifera. choice of the paternal side. Improved methods of instrumental bee insemination, however, have provided full control over the selection of the paternal side during mating. This significantly contributed to progress in bee breeding. Still, it is not known why instrumentally inseminated queen bees do not always commence oviposition. It is also not known why queens who have been instrumentally inseminated, start egg-laying later than naturallyinseminated queens (Mackensen, 1951, 1964; Jasi ski et al., 2005; Woyke et al., 2008).

6 In the past, the efficiency of properly performed instrumental insemination was affected by semen quality and lack of drone-specific diluents as well as other factors. Nowadays though, their analogues are applied with a common 0.9% saline solution and boar semen diluents. However, most diluents or substances are exposed to semen, reducing the survival rate of spermatozoa (Bishop, 1920). This explains why an air space in the insemination needle is necessary during the instrumental insemination. It prevents semen contact with the liquid in the needle and syringe. Another factor that affects the efficiency of the properly performed insemination procedure is semen competition. Inseminators claim that semen tends to coagulate in the needle during numerous insemination processes (Loc personal information). This coagulation markedly prolongs the insemination process. Internal complications may occur after natural mating between the queen bee and several drones (Schl ns et al., 2004; Andere et al., 2011). The internal complications may be caused by one drone, which, for instance, is the first to mate and contributes to semen coagulation. Semen coagulation consequently affects further sperm storage (Pizzari and Foster, 2008). After the mating, spermatozoa may compete between ejaculates from different drones (polyandry), which may induce their coagulation. The coagulation may be caused by incompatibility in the sex locus (Mackensen, 1964) as well as population selection. If this is so, none of the semen can be used since it remains in the queen bee s reproductive tract (Wojciechowski and Kr l, 1996). The survival of the semen stored by the queen bee is affected by the spermathecal fluid (Verma, 1973, 1978) and by substances secreted by the spermathecal glands (Schl ns et al., 2004). The spermatheca contains antioxidative enzymes protecting the spermatozoa from oxidative stress (Weirich et al., 2002). It is possible that such stress is induced by competition between the spermatozoa (Triasko, 1951). The competition involves increased motility, ensuring sperm precedence in reaching the queen bee s spermatheca. Another reason for lack of oviposition may be aggregation of spermatozoa from one drone, which form unbreakable bundles (Moritz et al., 1996). After copulation, the continually mixing spermatozoa are well visible through the transparent spermathecal wall (Ruttner, 1954; Woyke, 1960). After insemination with mixed semen originating from African and European drones, a higher rate of egg fertilization by African drones was observed. The ratio was 9:1 (Grandi-Hoffman et al., 2004; Schneider et al., 2006). Similar dominance was noted in wild drones and in those selected in the drone congregation areas with the progeny ratio 4:1, respectively (Woyke, 1971). This indicates high competition between spermatozoa (Tofilski et al., 2011). The introduction sequence of semen into a queen bee s reproductive tract also plays an important role. There is little probability of progeny from drones that were the last to be used for insemination (Woyke, 1963; Siuda et al., 2009). This is because excess semen from the queen bee s reproductive tract is removed. The techniques employed in the evaluation of the semen include the macroscopic method and the in vivo microscopic method. The macroscopic method uses the consistency estimation according to Woyke (1964). Woyke classifies semen into: 1) sparse semen, which is not suitable for insemination, 2) semen that is optimal for insemination, and 3) dense semen - also not suitable for insemination. Semen used in instrumental bee insemination can be assessed with the in vivo microscopic method based on the number of laid eggs (Jasi ski et al., 2005; Woyke et al., 2008). The aim of the experiment was to perform an in vitro and in vivo microscopic

Journal of Apicultural Science 7 analysis of diluted spermatozoa behaviour in diluents. Additionally, an attempt was made to determine the factors that may affect semen coagulation in the instrumental insemination process. MATERIALS AND METHODS The study was conducted in the Breeding Apiary in Teodor w, Poland. Carniolan honeybee Apis mellifera carnica drones and queen bees were investigated. The following semen smear groups were observed under the microscope: 1. according to drone age; Three age groups were distinguished: young drones up to 12 days old (sparse semen), mature drones which were from 14 to 27 days old (optimal semen for instrumental insemination), and older drones which were over 30 days old (dense semen); 2. in commercial diluents: saline, SAFE boar semen diluents; 3. 0.2 l semen with the spermathecal fluid from 4-6 virgin queen bees; 4. from partially filled spermatheca. The queen bees were inseminated by one drone with a dose of approximately 1 l. After commencement of oviposition the semen was collected from the spermatheca. Then, this semen was used for reinsemination of twelve virgin queens using a dose of approximately 0.5 l. The semen smears from the partially filled spermatheca and the spermatozoa after the reinsemination, were prepared to assess the behaviour of the spermatozoa in the spermatheca. The semen was mixed with diluents in a ratio of about 1:4. The smears were prepared on a B rker s plate. Twelve smears were made from each group. The smears were observed under an OLYMPUS microscope. The microscope was coupled with a digital camera to film the observed semen. Motility of spermatozoa/semen was observed in the preparations. A stopwatch was used to measure the length between the time the preparation was made and the beginning of spermatozoa/ semen movement - until full cessation of movement. The mean time of spermatozoa/semen movement in the preparations was calculated with SAS software (Institute, SAS User s Guide Version 9.1 ed., 2003) using univariate ANOVA. RESULTS AND DISCUSSION 1. Behaviour of the semen in relation to drone age Sparse semen from young drones had a cluster visible in loose spermatozoa bundles and short mobility duration (X SD=4.6 1.5 min, n=12) (Fig. 1). Similarly, Jaycox (1960), Lensky and Schindler (1967), Ruttner (1969) and Verma (1978) assessed the spermatozoa after cessation of movement. The optimal semen for instrumental insemination Fig. 1. Semen from a young drone.

8 Fig. 2. Semen from a mature drone. Fig. 3. Semen from an old drone. exhibited long mobility duration (X SD=26.6 2.3 min, n=12) and was accompanied by a high cluster mass of spermatozoa bonds (Fig. 2). Dense semen sampled from old drones displayed short mobility duration (X SD=15 5.7 min, n=12) in the microscopic picture (Fig. 3). This dense semen had a very high cluster mass of spermatozoa and varied mobility duration. Dense semen demonstrated high diversity and had a tendency to coagulate. The tendency to coagulate lowered the motility of the spermatozoa and induced their immobility. In the non-coagulated semen, movements of the spermatozoa were still visible. The mixed semen from two mature drones was very active but this activity later disappeared. Such reactions may indicate competition between the ejaculates (Triasko, 1951). Similar behaviour was observed in a mixture of ejaculates sampled from a young and a mature drone. At first, enhanced motility was observed. This enhanced motility may have been induced by an increased amount of the seminal vesicle fluid and of the endophallus bulb (Flanders, 1939; Woyke, 2008) or proteins (King et al., 2011). Sparse semen from immature drones did not coagulate when combined with the optimal consistency semen used

Journal of Apicultural Science 9 for insemination. The lack of semen coagulation may indicate that there is no hemolymph and endothelium in the young drone semen (Ruttner, 1954; Woyke, 2008, 2010). Dense semen caused rapid loss of motility and more frequent (44%) production of coagulates, not motility bands. The cessation of semen movement may have been induced by the increased amount of epithelium entering the semen (Woyke, 2008, 2010). Such an increase in epithelium may be a result of the drone aging process (Burzy ski, 2007; Rhodes et al., 2010; Paleolog et al., 2011). It may also result from the peeling of considerable amounts of the epithelium from mucus glands during eversion of the endophallus. 2. Behaviour of the semen in commercial diluents The semen from the young, mature and old drones was treated with a salinedilution. During the treatment, the spermatozoa displayed quick movements, which on average, ceased after X SD=12.0 6.2 min, n=12. Movement cessation was an indication of dying spermatozoa (Ruttner, 1969). Dilution resulted in disruption of the bonds and the first characteristic circular arrangement. Fig. 4. Semen from a mature drone mixed with the spermathecal uid of a non-inseminated queen bee (0,2 l semen with the spermathecal uid from 4-6 virgin queen bees). Fig. 5. Spermatozoa after reinsemination.

10 The boar semen diluent also induced increased spermatozoa motility accompanied by almost complete disruption of the bonds of the spermatozoa, which then restored the bonds. On average, during X SD=29.2 2.5 min, n=12 period, single spermatozoa at the sides of the preparations displayed the highest motility. 3. Behaviour of the semen in combination ith the spermathecal fluid Semen sampled from a mature drone and mixed with the spermathecal liquid of a non-inseminated queen bee initially displayed intensive motility. The mobility declined after a few minutes and remained at a constant level for average X SD=50.8 7.0 min, n=12 (Fig. 4). After the queen bees were inseminated with such a mixture and smears from their spermatheca were prepared, it appeared that the spermatozoa were arranged in loose bundles. These loose bundles formed circles which moved in a counter clockwise manner (Fig. 4). King et al. (2011) argue that sperm viability is dependent on protein and fructose found in seminal fluid whereas Gen er and Kahya (2011) state that sperm viability is dependent on lateral oviducts and spermathecae substances. 4. Behaviour of the semen sampled from the partially filled spermatheca In the partially filled spermatheca, highly motile spermatozoa arranged in the form of loose clusters were observed. Such spermatozoa have the ability of unconstrained movement and are not arranged in a cluster mass of spermatozoa bonds (Ruttner, 1954; Woyke, 2008, 2010), therefore their activity is increased. The higher the cluster of the spermatozoa in the spermatheca is, the more compact the bundles are. This means that the spermatozoa remain in tight clusters without being able to move. Characteristic behaviour was observed in the spermatozoa sampled from the spermatheca of the queen bees after reinsemination. The spermathecae were partially filled due to the small amount of semen sampled from a previously inseminated ovipositing queen. In all the smears, the spermatozoa formed more or less compact circles. The compactness depended on the amount of semen sampled from the spermatheca of an ovipositing queen bee (ca. 0.5 l). All the spermatozoa were arranged in circles, which displayed a rotary counter clockwise motion (all moving in the same direction) (Fig. 5). The circle arrangement may be related to the length of the spermatozoa, which is ca. mm (Ruttner, 1954). The circle arrangement facilitates storage of the long, circularly arranged spermatozoa in small, round, 1 mm in diameter spermathecae. The arrangement of the spermatozoa may also be related to polyandry. When sampled from several drones, the spermatozoa form circular clusters, mix in the queen bee s spermatheca (Ruttner, 1954; Woyke, 1960), and move into the vas deferens. Thus, the circular clusters close the vas deference temporarily until they become scarce. Other circular clusters then take their place. This is corroborated by the fact, that fertilized eggs laid by a queen bee recurrently descend from a different drone. A very visible example is in a bee family with naturally mated dark-coloured bees, where occasionally light-coloured bees appear after the queen bee has copulated with several light-coloured drones. CONCLUSION Semen from mature drones coagulates the earliest. Semen from young drones does not coagulate. ACKNOwLEDGEMENTS We thank Prof. Jerzy Woyke for critical support and constructive comments during the preparation of the manuscript. We also wish to thank Mr. Krzysztof Loc for his invaluable help in conducting the experiments.

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12 Pizzari T., Foster K. R. (2008) - Sperm sociality: Cooperation, altruizm, and spite. PLoS Biol. 6 (5): e 130. cloi; 10.137/journal. pbio.0060130. Rhodes J.W., Harden S., Spooner- Hart R., Anderson D. L., Wheen G. (2010) - Effects of age, season and genetics on semen and sperm production in Apis mellifera drones. Apidologie, 42(1): 29-38. Ruttner F. (1954) - Mehrfache Begattung der Bienenk nigin. Zool. Anz., 153: 99-105. Ruttner F. (1969) - The instrumental insemination of the queen bees. Apimondia Verlag, Bukarest, Romania. SAS Institute (2003) - SAS; Statistic s. SAS Institute Inc., Cary, NC, USA. Schl ns H., Koeniger G., Koeniger N., Moritz R. F. A. (2004) - Sperm utilization pattern in the honeybee (Apis mellifera). Behav. Ecol. Sociobiol., 56: 458-463. Schneider S. S., de Grandi-Hoffman G., Smith D., Tarpy D. (2006) - The African honey bee. I. A case study of a biological invasion. Bee Culture, 134(4): 21-24. Siuda M., B k B., Wilde J. (2009) - The impact of the sequence of particular drone semen administration on number of their own progeny after instrumental insemination Ann. Warsaw Univ. of Life Sc. - SGGW, Anim. Sci., 46: 109-113. Strassmann J. E. (2001) - The rarity of multiple mating by females in the social hymenoptera. Insectes Sociaux, 48(1): 1-13. Tofilski A., Chuda-Mickiewicz B., Czeko ska K., Chorobi ski P. (2011) - Flow cytometry evidence about sperm competition in honey bee (Apis mellifera). Apidologie, DOI: 10.1007/s13592-011-0089-6 Triasko W. W. (1951) - Priznaki osiemiennosti pczelinych matok [Signs ofmating of queen bees]. Pchelovodstvo, 28(11): 25-31. Verma L. R. (1973) - An ionic basis for a possible mechanism of sperm survival in the spermatheca of the queen honey bee (Apis mellifera L.). Comp. Biochem. and Physiol., 44(1): 1325-1331. Verma L. R. (1978) - Biology of honeybee (Apis mellifera L.) spermatozoa 1. Effect of different diluents on motility and survival. Apidologie, 9(3): 167-174. Weirich G., Collins, A., Williams V. (2002) - Antioxidant enzymes in the honey bee, Apis mellifera. Apidologie, 33(1): 3-14. Wojciechowski M., Kr l E. (1996) - On intraoviductal sperm competition in the honeybee (Apis mellifera). Folia Biol. Krakow, 44: 51-53. Woyke J. (1960) - Naturalne i sztuczne unasienianie matek pszczelich (Natural and artificial insemination of queen honey bees). Pszczeln. Zesz. Nauk., 4: 183-275. Woyke J. (1963) - Contribution of successive drones to the insemination of a queen. XIX-th Intern. Beekeeping Congress, Prague. Proceedings, Comp. Text. of Lect. of XIX Congress of Apimondia: 715-718. [online]: http://jerzy_woyke.users.sggw.pl/ contrsuccdr.pdf Woyke J. (1964) - Die Virkung aufeinanderfolgender Drohnen auf die Besamung der K nigin. Beekeep. Congr. Prague, 19: 550-552. Woyke J. (1971) - Unasienianie matek pszczelich na trutowisku o zwi kszonej liczbie trutni (The matings of honey bee queens in the mating station with enlarged drones numer). Pszczeln. Zesz. Nauk., 15(1-2): 43-51. Woyke J. (2008) - Why the eversion of the endophallus of honey bee drone stops at the partly everted stage and significance of this. Apidologie, 39(6): 627-636. Woyke J., Jasi ski Z., Prabucki J., Wilde J., Chuda-Mickiewicz B., Siuda M., Madras-Majewska B., Samborski J., Bratkowski J, Jojczyk A. (2008) - Onset of oviposition by honey bee queens, mated either naturally or by various instrumental insemination methods, fits a lognormal distribution. J. Apic. Res. and Bee World, 47(1): 1-9. Woyke J. (2010) - Three substances ejected by Apis mellifera drones during endophallus eversion as well as during natural matings with queen bees. Apidologie, 41(6): 613-621.

Journal of Apicultural Science 13 MIKROSKOPOwY OBRAZ PLEMNIK w TRUTNI PSZCZO Y MIODNEJ w TRZECH ROZCIE CZALNIKACH B o r s u k G., O l s z e s k i K., S t r a c h e ck a A., P a l e o l o g J., G a g o M. S t r e s z c z e n i e Celem bada by a mikroskopowa analiza zachowania si plemnik w w rozcie czalnikach in vitro i in vivo. Dodatkowo pr bowano okre li, kt re z czynnik w mog wp ywa na koagulacj nasienia podczas instrumentalnego unasieniania. Badano trutnie i matki rasy krai skiej Apis mellifera carnica. Utworzono cztery grupy do wiadczalne. Nasienie obserwowano w rozmazach mikroskopowych: 1. pobrane od trutni w r nym wieku. Wyodr bniono trzy grupy wiekowe: trutnie m ode - do 12-go dnia ycia (daj ce rzadkie nasienie), trutnie dojrza e - w przedziale wiekowym 14-27 dni (daj ce nasienie optymalne do instrumentalnego unasieniania), trutnie stare - powy ej 30-stu dni (daj ce g ste nasienie). 2. z handlowymi rozcie czalnikami (sol zjologiczn oraz z rozcie czalnikiem nasienia knur w SAFE), 3. z p ynem pochodz cym ze zbiorniczka nasiennego dziewiczych matek, 4. ze zbiorniczka nasiennego matki, kt r unasieniano jednym trutniem (dawka ok. 1 l) i czekano, a zacznie sk ada jaja nast pnie matk zabijano i pobierano nasienie ze zbiorniczka nasiennego, kt re wykorzystywano do powt rnego unasieniania dziewiczej matki - reinseminacja (dawka ok. 0,5 l). Rozmaz plemnik w ze zbiorniczka nasiennego matki, po reinseminacji u yto w celu stwierdzenia zachowania si plemnik w w zbiorniczku nasiennym innej dziewiczej matki. Nasienie mieszano z rozcie czalnikami w proporcji 1:4. Rozmazy wykonywano na p ytce B rkera. W ka dej grupie wykonano po 12 rozmaz w, kt re ogl dano w mikroskopie OLYMPUS. Mikroskop po czono z kamer cyfrow, kt r nagrywano lmy nasienia i wykonano zdj cia. W preparatach obserwowano ruch plemnik w/nasienia. Stoperem mierzono czas od wykonania preparatu, pocz tek ruchu plemnik w/nasienia w preparacie do ca kowitego zaprzestania ruchu. Nasienie od m odych trutni nie powodowa o koagulat w. Najcz ciej do koagulacji nasienia (44%) dochodzi o po zmieszaniu nasienia pochodz cego od starych trutni z nasieniem od dojrza ych trutni. Po 12 min obserwowano zaprzestanie ruchu plemnik w w nasieniu rozcie czonym sol zjologiczn. Plemniki w rozcie czalniku dla knur w wykazywa y oznaki ruchu przez 29 min. Plemniki po reinseminacji uk ada y si w ko a. S o a kluczo e: rozmaz nasienia, koagulacja nasienia, reinseminacja, trutnie, Apis mellifera.