Number of Spermatozoa in Spermatheca of Honey Bee (Apis Mellifera Carnica) Queens 0 and 1 Year Old Reared in the Conditions of Continental Croatia

ANIMAL HUSBANDRY agriculture in nature and environment protection

Izvorni znanstveni rad/original scientific paper

Number of spermatozoa in spermatheca of honey bee (Apis mellifera carnica) queens 0 and 1 year old reared in the conditions of continental Croatia

Marin Kovačić, Zlatko Puškadija, Ivor Šurlan Spitzmuller, David Kranjac

Faculty of Agriculture, Josip Juraj Strossmayer University of Osijek, Kralja Petra Svačića 1d, Osijek, Croatia, e-mail: [email protected]

Abstract One of the key factors for the success of honey bee colony is a productive queen. The queen mate in the air with several drones during one or more nuptial flights and store spermatozoa in the spermatheca. In our research we investigated the effect of the queens’ age on the number of spermatozoa and the diameter of spermatheca. In total, 137 queens were dissected, from which 71 young queens (Q0) and 66 queens that were in production colonies for one year (Q1). In the spermatheca of Q0 group, we found 3.96 million, and in Q1 group 2.64 million of spermatozoa in average, which is significant difference (p<0.05). Positive correlation was found between the queens’ weight and the size of spermatheca (r=0.45). Our results have shown that the number of sperm in spermatheca declines with the age of queen. Key words: Apis mellifera carnica, spermatheca, queen

Introduction Due to its proximity to man and high number of managed bee colonies, honey bees (Apis mellifera L.) are the most commonly studied social insects. Bee colony consists of fertile queen, facultative sterile worker bees and drones. Virgin queen is mated in the air on so called “drone congregation areas” during one or more nuptial flights (Schluns et al., 2004). Different results are reported regarding the number of drones queens mate with, so Taber and Wendel (1958) stated that queens mate with 7-10 drones, Neumann et al., (1999) reported 6-24, Kraus et al. (2005) 10-28 drones and Delaney et al. (2011) recorded even up to 50 drones . Nuptial flight of queens is key moment for colony and it should be effective and short (Koeniger and Koeniger, 2007) so that the risk of the queens’ loss is minimized. After mating, spermatozoa from oviduct enter spermatheca during next 40 hours (Woyke, 1983). Large number of stored spermatozoa is one of the basic conditions for high fecundity of the queen and thus high number of worker bees which will enable high productivity of colony. Some other features that characterise high quality queen are: high weigh of live queen, large number of ovarioles, size of spermatheca, high fecundity and disease-free (Hatjina et al., 2014). For rearing queens the age of larvae is one of crucial factors that affect queen morphology. The queens reared from newly hatched larvae are heavier and have bigger diameter of spermatheca than queens reared from older larvae (Gilley et al., 2003; Tarpy et al., 2000). Age of the queen affects productivity of colony and so far several studies have proved that colonies

257 9th international scientific/professional conference SECTION V with one year old queen produced more brood and more honey than colonies with older queens (Woyke, 1984; Akyol et al., 2007, Akyol et al., 2010, Simeunovic et al., 2014). During the peak of a season a queen lays 1500-2000 eggs per day and up to 12 spermatozoa enter each egg (Harbo, 1979). Quality of mating is highly dependent both on the density of drones in the area and weather conditions during nuptial flights, so warm and windless weather is needed for success of mating. In our research we wanted to see does the age of a queen affects the number of spermatozoa in spermatheca and is there a correlation between the age of queens, the diameter of spermatheca and the number of sperms.

Materials and methods In this research 0 and 1 year old mated queens were used for analysis. One year old queens (Q1) were provided by the beekeepers that replaced queens with new ones. Young queens (Q0) were taken from mating nucs 1 week after mating. After delivery all the queens were placed in freezer (-18oC) and kept untill dissection. Each queen was weight before dissection, using scale Kern Alj (max. 250g, min. 10 mg) with linearity ±0,3mg. Petri dish with wax was used for dissection. Spermatheca was isolated from queen with the tracheal net. The diameter was calculated in two distant points and the average of two measurements was taken. Dino Lite 7013 digital microscope camera was used for diameter measurement. Afterwards, spermatheca was crushed in 1 ml of saline solution and the number of sperm was counted using haemocytometer (Human et al., 2013). Statistical analysis was made in Statistica 12 software.

Results and discussion In total, 71 Q1 and 66 Q0 queens were dissected and on average 3.96 (±1.11) and 2.64 (±0.91) millions of spermatozoa were found in spermatheca, respectively (Table 1). Similar results are reported by Akyol et al. (2008), Gregorc et al. (2008), Al-Lawati et al. (2009), Tarpy et al. (2012), Hatjina et al. (2014), while some authors obtained different data (Haarmann et al., 2002; Lode- sani et al., 2004; Collins and Pettis, 2012; Gregorc and Smodiš, 2015). Table 1. Weight of queens, diameter of spermatheca and number of spermatozoa in spermatheca in queens aged 0 and 1 year.

Age of queen (years) N Weight (mg) Diameter of spermatheca Number of sperm (millions) (mm)

Q0 71 149,11 1,09 3,96

Q1 66 163,16 1,07 2,64 A positive correlation and significant difference was found between the queen age and the number of spermatozoa (r=0.54; p<0.05) (Graph 1.), and Q1 queens on average have 66.7 % of spermatozoa of 0 year old queens. It seems that older queens have spent 1/3 of spermathecal content in one year. If we consider increasing number of unviable spermatozoa in spermatheca (Lodesani et al., 2004), and slower movement of the sperm (Al-Lawati et al., 2009) with the queen aging, it is questionable wether such queen will enable adequate colony development in the third season. When we compare two groups of queens, it is noticeable that most of Q0 queens had between 3 and 5 million spermatozoa, while most of Q1 queens had between 2 and 3 million spermatozoa (Graph 2.).

258 ANIMAL HUSBANDRY agriculture in nature and environment protection

Dražić et al. (2015) have found more spermatozoa in spermatheca, and their measurements of diameter of spermatheca are almost the same, while Tarpy et al. (2012), who were checking quality of queen bees reared from the queen breeders in California, found on average 4,37 million. We found that Q0 queens have significantly more sperm stored than Q1 which is opposite to Dražić et al. (2015) who have found that 2 year old queens have more sperm than 1 year old queens.

Graph 1. Number of spermatozoa in spermatheca regarding the queen age

Graph 2. Number of spermatozoa in spermatheca in 0 year old queens (left) and 1 year old queens (right) A positive correlation was found between the weight of queens and the diameter- ofsper matheca (r=0.45, Graph 3.), which is similar to findings of Collins and Pettis (2012), and Akyol et al. (2008). The Q1 queens were heavier than Q0 (p<0,05) and this could be explained with fact that those queens were laying for much longer period and thus were fed with more royal jelly, while Q0 queens were laying eggs for only one week and they could not reach their po- tential body weight. Q1 queens were obtained from beekeepers. They were asked to store old, replaced queens in freezer and it is possible that beekeepers preserved best laying queens in the hives and exchanged only lower quality queens. This could be a reason that the number of spermatozoa in 1 year old queens in our study are similar to results of 2 year old queens from Lodesani et al. (2004), and Hatjina et al. (2014) who had 2.6 and 2.78 million respectively.

259 9th international scientific/professional conference SECTION V

Graph 3. Correlation of queen weight and diameter of spermatheca

Conclusion The number of spermatozoa in the spermatheca of queen bee decreases with aging, but well mated queen which stored more than 5 million sperm is capable of intensive oviposition of fertilized eggs for two years. Heavier queens have larger spermatheca that can receive more spermatozoa and thus can provide better colony development.

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