1 Vol. 55 No Journal of Apicultural Science 79 THE EFFECT OF DIAPAUSE TERMINATION TIME ON THE SUCCESSFUL INSEMINATION OF Osmia rufa L. FEMALES M o n i k a F l i s z k i e w i c z, K a r o l G i e j d a s z, Z d z i s ł a w W i l k a n i e c Department of Apidology, Institute of Zoology, Poznań University of Life Sciences, Wojska Polskiego 71C, Poznań, Poland. Received 22 July 2011; Accepted 26 September 2011 S u m m a r y One of the practical aspects of mason bee rearing is the need to synchronize bee activity with the flowering period of the target species. This means advancing or delaying the emergence relative to the natural activation term. The aim of the research was to determine the effect of reducing the duration of diapause of both males and females on insemination. Individuals activated earlier might, after mating and consequent nesting, be used in greenhouses for pollinating crops. Successful insemination is the prerequisite for obtaining a new generation. Therefore, there is a possibility that red mason bee females activated earlier in winter could give rise to two generations of offspring a year. Groups of red mason bees - 30 males and 30 females (in 3 replications) had diapause terminated in different months. After being removed from cocoons, they were placed in plexiglass isolation cages and kept in the laboratory with appropriate feeding and light conditions. After 14 days, the females were removed and killed, and the filling of their spermatheca was examined. Diapause was terminated in the first ten days of each November to March, and in April which is the month of the red mason bee s natural emergence and copulation time. The percentages of dead females to live (analysed) females and successfully inseminated to analysed females were compared with Chi-square contingency tests (procedure 'prop.test' from R package). Termination months differed significantly for both mortality and the successful insemination of Osmia rufa. Compared with natural activation in April, the most convenient month for terminating diapause was January, as differences in both mortality and insemination were not significantly different between these two months. Keywords: Osmia rufa L., diapauses, copulation, mating. INTRODUCTION Bees are the most important pollinators in many ecosystems and their activity is the main factor influencing yield in world agriculture (Batra, 1995). Honey bee (Apis mellifera L.) is the most important commercial pollinator, however, it is not sufficiently numerous before summer, and there are many flowers which the honey bee has difficulty accessing. Therefore, other mainly wild species of bees (Torchio, 1991; Kemp, 2000; Monzon et al., 2004; Komissar, 2005) such as Osmia rufa L. - the red mason bee, are of almost equal importance. Osmia rufa belongs to the Megachilidae family and is a common spring solitary bee species. Taking into account Osmia rufa biology, a method of artificial rearing has been developed, with the use of hollow plant stems (e.g. Phragmites australis - common reed) as nesting material. The stems are cut open in winter, and the cocoons with bees inside (as imago forms) are placed in a cold storage room, at 4-5 C (Wójtowski, 1979) and 40-50% humidity. Using this method of rearing, it is possible to synchronize (advance or delay),
2 80 to a certain extent, the active period of Osmia rufa to the flowering period of crop plants (Giejdasz and Wilkaniec, 2007). This method is also used in rearing other Megachilidae species (Pits-Singer et al., 2008; White et al., 2009). Mature individuals of Osmia rufa in the natural environment are already present in September, but they stay in cocoons during autumn and winter in a so-called winter dormancy (diapause). After wintering as imagos, they emerge from cocoons at the beginning of the orchard flowering period (Biliński and Teper, 2004). The main objective of the current research was to determine whether the bees whose diapause was terminated at different stages were able to copulate, and if so, what was the level of successful inseminations. Insemination of females is the prerequisite for obtaining a new generation of Osmia rufa. If the bees, after winter termination of diapause, begin mating and copulating, then inseminated females might be able to prepare nests for egg laying. This would raise the possibility of rearing a new generation of the red mason bee twice a year. Nesting activity involves supplying nest cells with pollen gathered from flowering plants. Many crops (e.g. strawberries) which are grown in greenhouses, bloom much earlier than when they are grown outdoors (Wilkaniec and Radajewska, 1997; Wilkaniec and Maciejewska, 1999). They make the price for the product much higher. Thus, there is already a demand for insect pollinators in winter time which Osmia rufa may be able to fulfil. Many greenhouse plants produce flowers in winter when there are no natural pollinators, so Osmia rufa bees which are active at this time, and able to copulate and lay eggs, might be used for pollinating greenhouse crop plants. MATERIAL AND METHODS The research was performed in the laboratory of the Department of Apidology, Institute of Zoology, Poznań University of Life Sciences, Poznań, Poland. Red mason bee mating in isolation cages The occupied nest tubes were transferred to the laboratory in October and were dismantled. Then, the adult bees in cocoons were removed from nest cells and placed in cold storage. Cocoons containing adult bees bred in the laboratory were removed from cold storage where they stayed during diapause, at 4 C and 40-50% humidity. Cocoons were carefully cut open lengthwise and the bees removed. On the basis of their sexual dimorphism, males and females were differentiated and separated from each other. Three replicate groups were formed for each month of termination, each consisting of 30 males and 30 females. Termination of bee diapause took place in each month from November to April- the natural time for red mason bee emergence and mating (Raw, 1972; Seidelmann, 1995). Diapause was terminated in the first ten days of each month. Bees were placed in plexiglass cuboid isolation cages (22 cm x 22 cm x 40 cm). Each cage had a round hole at the front with a tulle-made sleeve allowing researchers easy access to the inside. During the experiment, the hole was covered with a cardboard disc to prevent bees from entering the sleeve. Bees were kept in the isolation cages for two weeks to establish if they were mating. Bees were fed on pollen from ground honey bee pollen loads and on sugar-water syrup (1:1 ratio), served on 5 cm diameter Petri dishes. To prevent drowning, a perforated disc cut out of the comb foundation was placed on the dish, to enable bees safe and easy access to the syrup. The feed was provided ad libitum and changed daily. Heating lamps were placed over the isolation cages which were automatically switched on for 12 hours a day. The temperature in the laboratory was kept at 25 to 28 C.
3 Vol. 55 No Journal of Apicultural Science 81 The female spermathecae analysis After 14 days the females were taken from the isolation cages and killed with ethyl acetate. Their spermatheca were dissected and crushed, and afterwards the contents were thoroughly mixed with a 0.9% physiological salt solution. A drop of this solution was placed on the Fusch-Rosental camera and the presence of sperm cells was determined with the use of a phase contrast microscope (20 x 10 magnification). This method is typically used for examining the filling of the honey bee queen spermatheca (Woyke, 1966; Woyke and Jasiński, 1973). For the purpose of this research, the sperm cells were not counted, but their presence was verified. Moreover, the quantity of the physiological salt solution was modified compared to similar experiments conducted on honey bees. This procedure was done to check for successful insemination. As with the honey bee, the spermatheca of inseminated females were specifically shaped and coloured. They were milky white if there were sperm cells inside and transparent if a female was not inseminated. A total of 426 live females of Osmia rufa L. were examined. The percentages of dead females to live (analysed) females and successfully inseminated to analysed females were compared with Chi-square contingency tests, using the proportions test procedure in the R-package. RESULTS During the experiment, differences were observed in female mortality and in the rate of successful insemination. It proved impossible to examine the spermatheca of dead females to verify the presence of sperm cells. The number and the percentage of dead females, and of females successfully inseminated in different months are presented in Table 1. There were differences in the mortality of females whose diapause was terminated in different months (P<0.0052). The mortality of females, whose diapause had been terminated earlier (in November - March) was higher than the mortality of the females whose diapause was terminated in April (P<0.0081). However, there were some variations within the November - March mortality rates (P<0.0487). Relatively low mortality was observed in December and January, and the difference between those two months and April was not significant (P=0.1052; P=0.41, respectively). Thus, for Osmia rufa rearing and breeding, the months of diapause termination were not critical. The percentage of successfully inseminated females was different in particular months (P<0.0002). While comparing diapause termination in November-March to April, it was observed that April was the optimal time for successful insemination. The earlier diapause termination months were associated with a reduced insemination rate (P<0.0001). In November-March, the percentage of successfully inseminated females varied significantly (P<0.0333). Date of activation Numbers and proportions of dead, analysed and inseminated females in individual waking periods Number of settled females pcs. Number of dead females pcs. (%) Number of females subjected to further analysis, pcs. (%) T a b l e 1. Number of inseminated females, pcs. (%)* November (30) 63 (70) 11 (17.5) December (18.9) 73 (81.1) 21 (28.8) January (13.3) 78 (86.7) 32 (38.6) February (25.5) 67 (74.5) 17 (25.4) March (27.8) 65 (72.2) 22 (33.8) April (11.1) 80 (88.9) 42(52.5) * There was calculated the proportion of inseminated females to females subjected to further analysis
4 82 Since the mortality of Osmia rufa females in December and January was similar to that in April, the insemination rate in these months was also compared. In December, it was significantly lower than in April (P=0.0085), however in January the difference was not significant (P=0.09). Thus, the best time for diapause termination was January, since both mortality and successful insemination rate did not differ significantly compared to April. DISCUSSION The red mason bee development from egg to adult takes approximately 100 days (Giejdasz and Wilkaniec, 2002). From September onwards, individuals in cocoons are fully developed and pass the winter dormancy period as imago forms. For many insect species, diapause is connected with a temporary shortage or reduced availability of food (Danks, 1987; Alekseev, 1990). Many bee (Apoidea) species, whose lifespan exceeds one vegetative season, have adapted to harsh conditions by developing a form capable of going through winter diapause. The current research proved it is possible to early terminate red mason bee diapause, although at the expense of a successful insemination rate. Seidelmann (1999) stated the male spermathogenesis process must be finished in spring, by the time of emergence of the females. Similarly, Thalmann (1991) confirms that Osmia rufa male spermathogenesis completion occurs at the moment of their emergence. This research showed that males whose diapause was precociously terminated are still able to produce semen, since the females were successfully inseminated. However, when using Osmia rufa for winter and early spring pollination, it would be advisable to increase the bee stock, since the mortality and the rate of non-inseminated females is higher than at their natural time of emergence. In our study, the maximum survival (defined two weeks after diapause was terminated) was obtained in April. The survival rate of red mason bee females under laboratory conditions was not less than 90% in the three week period, when bees emerged in April (Giejdasz and Wilkaniec, 2007). Survival of Osmia cornuta females increased sharply when bees were wintered for 30 days or more and the maximum survival rate (70%) was obtained in February (Bosch and Kemp, 2004). Longevity of females kept without feeding at 20ºC was influenced by wintering duration wintering duration (Bosch and Kemp, 2004). In our study, date of diapause termination had an effect on both mortality and successful insemination rate, which fluctuated parallel to each other. It seems that in bee groups with high mortality most of the females that survived the two week evaluating period were not vigorous enough to copulate. Many crops (e.g. strawberries) which are grown in greenhouses bloom much earlier than outdoor grown crops (Wilkaniec and Radajewska, 1997; Wilkaniec and Maciejewska, 1999). Such greenhouse grown crops make the price for the product much higher. Thus, there is already a demand for insect pollinators in winter time which Osmia rufa may be able to fulfil. CONCLUSIONS The timing of the red mason bee (Osmia rufa L.) diapause termination influences both mortality and successful insemination rate. The survival and the rate of inseminated females is higher than at their natural time of emergence. Many crops, which are grown in greenhouses bloom much earlier than outdoors. Such greenhouse grown crops make the price for the products much higher. We recommend that Osmia rufa fulfil this early demand for insect pollinators in winter time. Acknowledgments The authors is very grateful to Prof. dr. Piotr Tryjanowski of the Zoology Institute, Poznań University of Life Sciences for his helpful remarks and support.
5 Vol. 55 No Journal of Apicultural Science 83 REFERENCES Alekseev V. R. (1990) - Diapauza u rakoobraznykh: ekologo-phyziologitcheskije aspekty. Nauka Press, Moskva. Biliński M., Teper D. (2004) - Rearing and utilization of the red mason bee - Osmia rufa L. (Hymenoptera,Megachilidae) for orchard pollination. J. Apic. Sci., 28(2): Batra S. (1995) - Bees and pollination in our changing environment. Apidologie, 26: Bosch J., Kemp W. P. (2004) - Effect of pre-wintering and wintering temperature regimes on weight loss, survival, and emergence time in the mason bee Osmia cornuta (Hymenoptera: Megachilidae). Apidologie, 35: Danks H. V. (1987) - Insect dormancy: an ecological perspective - Biological Survey of Canada. Monogr. Ser. 1, National Museum of Natural Sciences, Ottawa. Giejdasz K., Wilkaniec Z. (2002) - Individual development of the red mason bee (Osmia rufa L., Megachildae) under natural and laboratory conditions. J. Apic. Sci., 46(1): Giejdasz K., Wilkaniec Z. (2007) - Wpływ terminu aktywacji pszczoły murarki ogrodowej Osmia rufa L. na przeżywalność samic. [The effect of red mason bee activation time on female longevity]. XLIV Apicultural Scientific Conference, Puławy, th of April: Kemp W. P. (2000) - The future of crop pollination. Am. Bee J., 140(11): Komissar A. (2005) - Rearing osmia bees in Ukraine. The Beekeepers Quarterly, 82: Monzón V. H., Bosch J., Retana J. (2004) - Foraging behavior and pollinating effectiveness of Osmia cornuta (Hymenoptera: Megachilidae) and Apis mellifera (Hymenoptera: Apidae) on Comice pear. Apidologie, 35(6): Pits-Singer T. L., Bosch J., Kemp W. P., Trostle G. L. (2008) - Field use of an incubation box for improved emergence timing of Osmia lignaria populations used for orchard pollination. Apidologie, 39: Raw A. (1972) - The biology of the solitary bee Osmia rufa (L.) (Megachilidae). Transactions of the Royal Entomological Society of London, 124: Seidelmann K. (1995) - Untersuchungen zur Reproduktionsbiologie der Roten Mauerbiene, Osmia rufa (L., 1758), Dissertation, Univ. Halle. Seidelmann K. (1999) - The Race for Females: The Mating System of the Red Mason Bee, Osmia rufa (L.) (Hymenoptera: Megachilidae). J. Insect. Behav., 12: Tasei J. N. (1973) - Le comportement de nidification chez Osmia cornuta Latr. et Osmia rufa L. (Hymenoptera Megachilidae). Apidologie 4: Torchio P. F. (1991) - Bees as crop pollinators and the role of solitary species in changing environments. Acta Horticulturae, 288: Thalmann U. (1991) - Ausgewählte Untersuchungen zur Reproduktionsbiologie der Solitärbienen-Arten Anthophora acervorum (Linné, 1758) und Osmia rufa (Linné, 1758). Dissertation, University Press, Cambridge, MA. Wilkaniec Z., Maciejewska M. (1999) - Pollination of strawberry by solitary bee Osmia rufa L. (Apoide, Megachilidae ) and its effect on the yielding and quality of fruits. Rocz. AR w Poznaniu, CCCXIX, Zootech. 51: Wilkaniec W, Radajewska B. (1997) - Solitary bee Osmia rufa L. (Apoidea, Megachilidae) as pollinator of strawberry cultivated in an unheated plastic tunnel. ISHS Acta Horticulturae 439: III International Strawberry Symposium, 1 September Veldhoven, Netherlands.
6 84 White J., Son Y., Park Y. (2009) - Temperature-dependent emergence of Osmia cornifrons (Hymenoptera: Megachilidae) adults. Journal of Economic Entomology, 102: Woyke J. (1966) - Wovon hänht die Zahl der Spermien in Samenblase der auf naturlichem Wege begatteten Königinen ab? Zeitschr, Für Bienenforsch. 8(3): Woyke J., Jasiński Z. (1973) - Influence of external conditions and the number of spermatozoa entering the spermatheca of instrumentally inseminated honeybee queens. J. Apic. Res., 12: Wójtowski F. (1979) - Spostrzeżenia nad biologią i możliwościami użytkowania pszczoły murarki - Osmia rufa L. (Apoidea, Megachilidae). [Remarks on the biology and use of the red mason bee - Osmia rufa L. (Apoidea, Megachilidae)]. Rocz. AR w Poznaniu, CXI : Wpływ długości okresu diapauzy na stopień unasienienia samic murarki ogrodowej (Osmia rufa L.) F l i s z k i e w i c z M., G i e j d a s z K., W i l k a n i e c Z. S t r e s z c z e n i e Jednym z praktycznych aspektów chowu murarki ogrodowej z wykorzystaniem sztucznych gniazd jest możliwość zsynchronizowania terminu aktywacji pszczół z terminem zakwitania upraw, które mają być przez nie zapylane. Oznacza to przyspieszanie bądź opóźnianie terminu aktywacji pszczół w stosunku do terminu ich naturalnego pojawu. Celem doświadczenia było określenie wpływu terminu wybudzania owadów (samic i samców) w okresie diapauzy (przyspieszanie aktywacji) na stopień unasienienia samic. Wybudzane wcześniej osobniki murarki, które przystąpią do kopulacji, a w konsekwencji tego do zakładania gniazd można będzie wykorzystać do zapylania upraw szklarniowych. Unasienienie samic jest głównym warunkiem uzyskania pokolenia potomnego. Być może samice unasienione w miesiącach zimowych po zbudowaniu przez nie gniazd i złożeniu jaj pozwoliłyby na uzyskanie drugiego pokolenia w roku. Grupy owadów - 30 samic i 30 samców (w trzech powtórzeniach) wybudzano w różnych terminach trwania diapauzy i po wyjęciu z oprzędów umieszczano w klatkach hodowlanych wykonanych z pleksiglasu. Klatki hodowlane przetrzymywano w laboratorium, gdzie zapewniono owadom odpowiednie warunki świetlne i żywieniowe. Po 14 dniach od umieszczenia owadów w izolatorach wyjmowano samice, usypiano je i przeprowadzano analizę wypełnienia ich zbiorniczków nasiennych. Terminami wybudzania owadów były pierwsze dekady: listopada, grudnia, stycznia, lutego, marca i kwietnia - termin kontrolny (czas naturalnego wygryzania się murarki ogrodowej z oprzędów i odbywania kopulacji). Odsetek samic martwych i samic unasienionych porównano testem Chi-kwadrat, a także użyto testu R-package. Termin wybudzania O. rufa L. (skracanie długości diapauzy) ma istotny wpływ zarówno na śmiertelność, jak i na stopień unasienienia samic. Poza terminem naturalnej aktywacji O. rufa L. (kwiecień), styczeń jest najdogodniejszym terminem wybudzania tego gatunku, gdyż zarówno śmiertelność, jak i stopień unasienienia nie różnią się statystycznie w stosunku do terminu kwietniowego. Słowa kluczowe: Osmia rufa L., diapauza, kopulacja, unasienianie.