Journal of Apicultural Research 43(4): 167–171 (2004)
© IBRA 2004
ORIGINAL ARTICLE
Functional longevity of honey bee, Apis mellifera, queens inseminated with low viability semen ANITA M COLLINS USDA-ARS, Bee Research Laboratory, 10300 Baltimore Ave., Bldg. 476, BARC-East, Beltsville, MD 20705, USA Received 5 January 2004, accepted subject to revision 5 February 2004, accepted for publication 13 August 2004
SUMMARY Queen honey bees artificially inseminated using semen with 46% or more live spermatozoa consistently laid all fertilized eggs in normal worker brood patterns at 3–4 weeks after insemination. However, queen producers that might use stored semen to preserve breeder stock would want to rear daughter queens throughout a season, longer than these experimental queens were observed. To determine how long queens inseminated with low viability semen continue to produce worker brood, sister queens were inseminated with various mixtures of fresh and freeze-killed semen, or were allowed to mate naturally. Colonies were evaluated for area of comb with all stages of brood, the percentage of worker vs. drone offspring in sealed brood, and the number of empty cells in a representative area of sealed brood once a month for 5–6 months, at midwinter, and again the following spring. One queen of 22 queens inseminated with 75% or more fresh semen became a partial drone layer by mid winter, only two of the 21 queens inseminated with half fresh semen ran out of sperm, but 3 of 5 of queens with 25% fresh semen were laying some proportion of unfertilized eggs. Preserved semen that has 50% or better viable sperm can be used to inseminate queens that will function as well as a fully mated queen for at least one season, such that a breeder could rear many daughter queens and incorporate desirable genotypes into a breeding programme. Keywords: spermatozoa, instrumental insemination, artificial insemination, drone brood, fertilization, viability, germplasm preservation, freezing semen
INTRODUCTION The value of a technology for preserving honey bee semen depends on whether stored semen can be used successfully in artificial insemination of queens. Commercial use of stored semen will probably involve insemination of breeder queens representing a specific stock from which many daughter queens will be reared. In common practice, a breeder queen would be used for production of queens during a single season (spring to late summer). Experience has shown that if queens receive a sufficient volume (8 µl) of fresh semen they will head production colonies as well as naturally mated queens (Laidlaw & Page, 1997). Low levels of spermatozoa in the spermatheca (normal ranges are 3 to 7 million (Mackensen & Roberts, 1948)) will result in unfertilized eggs (drones) being laid in worker brood areas, a condition known as ‘drone laying’. This condition can occur from poor mating (few drones and small volume of semen) or from the depletion of spermatozoa due to extensive egg production. From the queen breeder’s viewpoint, a drone-laying queen is a poor choice for grafting, as drone larvae are not acceptable for rearing queens. Initial experimentation with liquid nitrogen storage of semen by Harbo (1977, 1979), Kaftanoglu & Peng (1984) and others (Melnichenkov & Vavilov, 1975, Verma, 1983) had limited success because a high proportion of the spermatozoa were killed or damaged in the process. Based on evaluation of the occurrence of drones in worker brood from inseminated queens, Harbo estimated that he had 20–25% spermatozoa survive. Kaftanoglu & Peng had 47.3% worker progeny. More recently using mixes of freeze-killed and fresh semen, I determined the level of viability of spermatozoa necessary to produce a good brood pattern from an inseminated queen to be 46% live or better *Corresponding author:
[email protected]
(Collins, 2000a). Queens inseminated using semen with less than 46% live spermatozoa had a high probability of being drone layers and stored fewer spermatozoa in the spermatheca. Viability of the spermatozoa was measured directly by dual fluorescent staining with propidium iodide and SYBR-14 (Collins & Donoghue, 1999). However, the queens mentioned above (Collins, 2000) were dissected at 24–25 days after insemination to determine levels of live spermatozoa present in the spermathecae. Because dissection abruptly terminated any further egg laying, there was no opportunity to evaluate how long an acceptable level of egg fertilization might continue. No studies designed to determine the levels of viable sperm in drone-laying queens have been reported. It is possible that the queens that were inseminated with lower levels of viable sperm might have become drone layers rather quickly if they had been allowed to continue laying eggs. Presented here are the results of an experiment to determine how long honey bee queens inseminated with mixtures of fresh (live) and frozen (dead) spermatozoa will continue to lay all fertilized eggs in worker cells and produce sufficient worker brood to sustain a colony.
MATERIALS AND METHODS In spring 2002 and 2003, 30 equalized, one-story colonies were started in a single apiary in Beltsville, MD, USA. Once the colonies were well established, the resident queens were removed and 24 h later mature queen cells from one breeder source introduced. Additional queens were emerged in an incubator, held in cages in a queen bank and used to replace missing queens as needed.
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Key to figures 1 and 2 Normal Supercedure Swarmed Drone layer Poor brood Diseased brood Killed
2002
Colonies
Control
95%
Half fresh
90%* 5%
Quarter fresh
5%
Jul 3
Jul 29
Aug 20
Oct 2
Date Sept 10
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Mar 8
FIG. 1. Each bar represents the functional life of a queen. Shading/pattern of the line indicates the reason for failure or removal at the time of failure or loss. March losses were winter colony deaths. * This queen began as a drone layer, shifted to all worker brood, and then became a drone layer again during the winter.
2003
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Jun 24
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FIG. 2. Each bar represents the functional life of a queen. Shading/pattern of the line indicates the reason for failure or removal at the time of failure or loss. March losses are colony deaths over winter.
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Queen’s longevity and semen viability When virgin queens were one- to two-weeks old, they were artificially inseminated (Harbo, 1985) with (1) all fresh semen, (2) 75% semen : 25% frozen semen, (3) 50% fresh : 50% frozen semen, or (4) 25% fresh semen : 75% frozen semen. Treatment groups included unequal numbers of queens, as I was most interested in the longevity of half fresh inseminations based on previous results discussed above (Collins, 2000a). Semen was collected from mature drones of any local source in the usual manner using a Harbo syringe fitted with 40 µl glass capillary storage tubes (Harbo, 1974). In 2002, several tubes of semen were frozen at –20 °C overnight to kill all of the spermatozoa. At the time of artificial insemination (AI), fresh semen was collected directly from mature drones to the desired volume in the syringe tip, and then the remaining amount to a total of 8 µl was collected from thawed frozen semen expelled into an Eppendorf tube. The semen used in 2003 was collected in the same manner, but all of it was stored in capillary tubes until used for AI. Half the tubes were held in the freezer as before; the other half of the collection was held in sealed tubes at room temperature for 1–3 d – storage conditions unlikely to reduce viability (Collins, 2000b). Mixtures of semen for the inseminations were made by drawing measured volumes of fresh and/or frozen semen into a calibrated syringe tip to a total of 8 µl. As a prophylactic measure, one µl of 0.1% saline with streptomycin (Harbo, 1985) was introduced with all of the inseminations. After insemination, queens had one wing clipped to prevent flying and a coloured, numbered tag glued to the thorax for identification. The following day they were narcotized with CO2 a second time. Additional queens of the same stock (2002) or sisters of the inseminated queens (2003) were allowed to mate from their natal colony. Once these naturally mated queens were observed to be laying eggs, one wing was clipped and the thorax marked. The naturally mated and all-fresh inseminated queens were the experimental controls. Several weeks after insemination the queen was located in each colony. If the correct marked queen was present, the area of all brood was estimated using a Plexiglas sheet marked in 5 x 5 cm squares (25 cm2). The number of squares of eggs, larvae and sealed pupae on both sides of each comb was measured to the nearest half square. A template on the Plexiglas was also marked to outline a square of 100 cells, and the number of empty cells in a representative area of 100 sealed worker cells counted. This was used as an indicator of the quality of the egg laying pattern. For those queens showing signs of becoming drone layers, the percentage of drone cells found in a representative area of sealed brood in worker cells was counted. Queen verification and all measurements were repeated every 3–4 weeks until the queen was superseded or the weather prohibited colony manipulations. In 2002, observations were made on 3 July, 29 July, 30 August, 10 September and 2 October. In 2003 the observation dates were 24 June, 24 July, 19 August, 24 September, 2 October and 7 November. I was also able to check the queens each March (winter). The following springs (April) the queens were located a final time and the experiment terminated. Queens were missing for a variety of reasons. In this study supersedure indicates both an early queen loss (before the first observation) which might have been due to injury or infection from the AI process, and later losses often with queen cells found on the combs. In several cases I chose to terminate the queen because she was laying few eggs or mostly drones, or the colony was badly diseased (chalkbrood). The numbers of queens surviving in each treatment within year were compared by Chi-square analysis. A Student’s t test was used to verify equality of the mean number of empty cells in a patch of 100. For each year, the low viability treatments were compared to the control, and the overall means of the two years compared. The brood areas were analysed by analysis of variance (PROC GLM, SAS 2000) with a model including date, treatment and the interaction.
RESULTS In 2002, about one-third of the queens did not survive to insemination. Also early in the season, two queens were lost accidentally, three colonies were robbed of all their honey, and one swarmed. The limited observations on these queens were not included in the analysis. One queen was accidentally lost in a move to a new colony late in the season, but her observations were included with the data. Observations are reported from a total of 14 control queens, eight queens from the 75% fresh treatment, 21 from the 50% fresh group, and five inseminated with 25% fresh semen. Figures 1 and 2 outline the fate of queens during the experiment for 2002 and 2003, respectively. Losses due to supersedure occurred in all groups but 25% fresh, and were not different between treatments (χ2 = 3.24, 16 df, P = 0.999 for 2002; χ2 = 0.524, 10 df, P = 1.00 for 2003). One queen of the 14 controls (7%) laid unfertilized eggs (drones) in worker cells, and she did not begin laying drones until winter. No queens of the 75% fresh treatment were drone layers. The 50% fresh group included one queen that was a drone layer (95% of the pupae) early in the season and one (combining to 9.5% of the group) that failed in winter (100% drones). The group inseminated with the least viable semen 25% fresh) had one drone layer (20% of queens; 90% drone pupae) and two (40%) that consistently had a few drones (5%) scattered within a patch of worker brood, evidence that these queens had insufficient spermatozoa and were laying unfertilized eggs. The areas of brood produced by the normally laying queens were not significantly different between the semen treatments (F = 1.55, 2 df, P = 0.224 for 2002; F = 1.08, 2 df, P = 0.346 for 2003). Figures 3 and 4 show the seasonal patterns for all colonies in each treatment group for 2002 and 2003, respectively. There were significant differences between observation periods in 2003 (F = 29.99, 4 df, P < 0.0001). Brood production peaked in August and then declined to a significantly lower level in the late autumn (November observation). The occurrence of empty cells within a patch of sealed worker brood was not significantly different for the treatments within each year (table 1) (all t < 1.0, df = 9,7,13,8, all P > 0.24). However, the overall means of the two years were significantly different (t = 6.36, 33 df, P < 0.0001).
DISCUSSION The results of the inseminations from this experiment were consistent with those of the previous study (Collins, 2000a) in that only the queens that received less than 50% fresh semen had a high probability of laying unfertilized eggs in worker comb. The rest of the queens performed as well as fully inseminated queens throughout the beekeeping season, producing solid patterns of worker brood in worker comb, although there was variation in area of brood from queen to queen. In fact, one queen inseminated with 50% fresh semen was laying so well that I left her in the colony TABLE 1. The number of empty cells (± standard deviation) in a set of 100 cells randomly chosen in a representative area of sealed brood. ** The overall means of the two years are significantly different with t = 5.6, 16 df, P < 0.0001. Treatment
2002
2003
Control
332.5 ± 7.2
11.5 ± 4.3 11.1 ± 2.1
3/4 fresh 1/2 fresh
40.1 ± 22.1
12.8 ± 5.2
1/4 fresh
29.0 ± 8.9
–
Mean**
34.6 ± 15.1
12.1 ± 4.2
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Controls
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FIG. 3. The area (cm2) of brood measured in each colony of the three treatment groups (controls = all fresh semen for artificial insemination or naturally mated; 50% fresh : 50% frozen semen, 25% fresh : 75% frozen semen) that survived in 2002. Drone layers: squares in 50% and 25% graphs; weak = a colony that had a small population all season.
FIG. 4. The area (cm2) of brood measured in each colony of the three treatment groups (controls = all fresh semen for artificial insemination or naturally mated, 75% fresh : 25% frozen semen, 50% fresh : 50% frozen semen) that survived in 2003. The values for August and November were significantly different from the other months (F = 29.99, 4 df, P
