CSIRO PUBLISHING
Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD11088
Sequential generations of honey bee (Apis mellifera) queens produced using cryopreserved semen Brandon K. HopkinsA,C, Charles HerrB and Walter S. Sheppard A A
Washington State University, Department of Entomology, Pullman, WA 99164-6382, USA. Center for Animals Near Biological Extinction, 1016 S. Main Street, Colfax, WA 99111, USA. C Corresponding author. Email:
[email protected] B
Abstract. Much of the world’s food production is dependent on honey bees for pollination, and expanding food production will further increase the demand for managed pollination services. Apiculturists outside the native range of the honey bee, in the Americas, Australia and eastern Asia, have used only a few of the 27 described subspecies of honey bees (Apis mellifera) for beekeeping purposes. Within the endemic ranges of a particular subspecies, hybridisation can threaten native subspecies when local beekeepers import and propagate non-native honey bees. For many threatened species, cryopreserved germplasm can provide a resource for the preservation of diversity and recovery of endangered populations. However, although instrumental insemination of queen honey bees is well established, the absence of an effective means to cryopreserve honey bee semen has limited the success of efforts to preserve genetic diversity within the species or to develop repositories of honey bee germplasm for breeding purposes. Herein we report that some queens inseminated with cryopreserved semen were capable of producing a substantial number of fertilised offspring. These diploid female larvae were used to produce two additional sequential generations of new queens, which were then back-crossed to the same stock of frozen semen. Our results demonstrate the ability to produce queens using cryopreserved honey bee spermatozoa and the potential for the establishment of a honey bee genetic repository. Additional keywords: conservation, instrumental insemination. Received 9 April 2011, accepted 9 February 2012, published online 10 April 2012
Introduction Populations of managed honey bees have declined in the US for approximately 50 years, due, in part, to demographic changes in the beekeeper population, the economics of the industry and increased losses due to introduced parasites (Stokstad 2007). Recently, numerous beekeepers worldwide have reported even higher rates of colony loss, ranging from 35%–75% annually, a condition termed ‘colony collapse disorder’ (CCD; Stokstad 2007; Underwood and vanEngelsdorp 2007; vanEngelsdorp et al. 2010). One recent review suggested that at least 61 factors could be involved in CCD, including pathogens, nutrition, moving stress and pesticide exposure (vanEngelsdorp et al. 2009). Large declines in managed populations of Apis mellifera in the US and elsewhere pose a serious threat to the underlying genetic diversity of these stocks. The genetic diversity of the species is further threatened by the potential loss of some of the 27 described subspecies of A. mellifera found in Europe, Africa and Asia (Sheppard and Meixner 2003). Transhumance of honey bees and utilisation of only several subspecies for commercial management has been further detrimental to native subspecies in some locations through fragmentation, hybridisation and selective breeding (Soland-Reckeweg et al. 2009). Journal compilation ! CSIRO 2012
The United Nations Food and Agriculture Organisation (FAO) developed the Interlaken Declaration and Global Plan of Action (GPA), detailing conservation measures for agricultural animal genetic resources. Part of the GPA calls for the formation of a cryogenic gene bank containing all livestock species (FAO 2007). Honey bees are a critical component of food production and are a valuable agricultural species. However, the GPA has not outlined a strategy for the preservation of honey bee genetic diversity. This may be due, in part, to the difficulty and expense required to establish a repository of honey bee genetic diversity through sustained management of live honey bee colonies. Liquid nitrogen storage of gametes provides a means for the long-term storage of genetic material. Cryopreservation of semen has been used to maintain valuable breeding material for several animal species of agricultural importance (Blackburn 2009). The ability to store honey bee genetic material for later use would make it possible to maintain or increase genetic diversity in selected honey bee stocks. Higher intracolony diversity has been shown to increase disease resistance and reduce parasite loads in honey bees (Tarpy 2003; Tarpy and Seeley 2006; Mattila and Seeley 2007). Even in light of drastic www.publish.csiro.au/journals/rfd
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honey bee population declines in many areas, the collection and preservation of genetic material from throughout the range of the honey bee would represent a major resource for continued selection and breeding in this agriculturally important beneficial insect. Honey bees are haplodiploid organisms, with females (workers) resulting from fertilised (diploid) eggs and males (drones) resulting from unfertilised (haploid) eggs, and normally a large percentage of workers is required to maintain a functional colony. Previous attempts at instrumental insemination (II) in honey bees using cryopreserved semen reported insufficient worker-to-drone ratios for the maintenance of a functional hive (Harbo 1979, 1983; Kaftanoglu and Peng 1984). However, from the standpoint of maintaining a genetic resource, successful cryopreservation in honey bees should not be measured by the ability to sustain functional hives with II queens, but rather the ability of such queens to produce enough diploid offspring to produce viable daughter queens. In a previous study, Harbo (1981) reported that queens inseminated with frozen semen produced inadequately viable embryos and, therefore, discontinued the development of semen cryopreservation. Recent work has been published on the development of cryopreservation methods for honey bee semen, but this did not use II of queens as an assay, but rather staining of live and/or dead cells to determine semen viability after thawing (Taylor et al. 2009; Hopkins and Herr 2010). Herein, we report on the use of an improved honey bee semen cryopreservation methodology (Hopkins and Herr 2010) for the II of honey bee queens and demonstrate its effectiveness in the production of successive generations of queens from a stock of cryopreserved semen. We discuss the suitability of this methodology for the establishment of honey bee germplasm repositories for conservation and breeding purposes. Materials and methods Semen collection Semen was collected from approximately 100 adult honey bee drones using a Harbo syringe (Harbo 1985). The semen used in this experiment was mixed 5 : 1 with semen extender diluent. The composition of the semen extender used in the present study (in a final volume of 100 mL) was: penicillin 0.0125 g; streptomycin 0.011 g; kanomyocin 0.015 g; tylosin 0.8 mg; N-[tris(hydroxymethyl)methyl]- 2-aminoethanesulfonic acid (TES) buffer 30 mM; Tris base 30 mM; EDTA 0.01 mM; sodium phosphate dibasic 1 mM, sodium citrate 1 mM; sucrose 0.5 mM; trehalose 0.5 mM; glucose 2.7 mM; arginine 0.57 mM; glycine 0.1 mM; proline 4.3 mM; catalase 0.5 mg; bovine serum albumin (BSA) 2 mg; KCl 82 mM; NaCl 83 mM; NaHCO3 5 mM. All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). After collection, the semen was held until use in a glass capillary tube, sealed with petroleum jelly and stored at room temperature in an insulated box. Semen was collected and frozen on the same day. Cryopreservation diluent The cryopreservation diluent included three components: buffer, cryoprotectant and chicken egg yolk. The diluent was
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Fig. 1. Diagram representing how semen straws were filled. From left to right the section are as follows: cotton, gelatin, cotton, extender solution, air space, semen, air space, extender solution, air space. The straw is then heat sealed at the end.
prepared with 500 mL buffer, 250 mL dimethyl sulfoxide (DMSO; D-8418; Sigma-Aldrich), and 250 mL egg yolk mixed fresh on the day of use. The buffer consisted of 79.7 mM NaH2PO4 (catalogue no. S-8282; Sigma-Aldrich) and 31.6 mM Na2HPO4 (catalogue no. S-7907; Sigma-Aldrich) made up to a final volume of 25 mL using water purified to 18.6 MO and pH adjusted to 7.2 using 6 M NaOH. This diluent is referred to hereafter as Harbo’s diluent. In the initial experiment we tested an egg yolk-free diluent that was formulated as above except that 250 mL of an egg yolkreplacement solution was used instead of 250 mL egg yolk. The solution used in place of the egg yolk was formulated by adding 20 mL of a 10% w/v cholesterol solution dissolved in 95% ethanol to 10 mL of 20% w/v BSA solution (catalogue no. 11020–021; Invitrogen, Carlsbad, CA, USA) in Schneider’s Insect Medium (catalogue no. S9895; Sigma-Aldrich). An alternative to egg yolk was tested because egg yolk has the potential to contaminate semen and the composition is not always uniform (Bergeron and Manjunath 2006). Mixing semen and diluent Immediately after collection, semen was mixed with extender in a ratio of 5 : 1. The semen–extender mixture and the cryopreservation diluent were then combined in a ratio of 3 : 2 by directly expelling the semen from the capillary tube used for collection with a Harbo syringe backfilled with Fluorinert (catalogue no. F4758; Sigma-Aldrich) into a 200-mL centrifuge tube. The diluent was then pipetted onto the semen in the centrifuge tube and the plastic tip of the pipette was used to gently mix the semen and diluent. Freezing containers The semen–diluent mixture was loaded into 0.25-mL Cassou straws (Edwards Agri-Sales, Menomonie, WI, USA) cut to a length of 6.5 cm, including the cotton/gelatin plug. Straws were loaded with 20 mL extender, an air space, 20 mL semen–diluent mixture, air space and approximately 20 mL extender or until the initial fluid sealed the plug (Fig. 1). The open ends of the straws were sealed with heated forceps. Slow cooling and cryopreservation Straws loaded with semen–diluent mixture were placed in a plastic queen cage (catalogue no. 508; Brushy Mountain, Moravian Falls, NC, USA). The cage containing the straws was submerged vertically in 450 mL room temperature water in a 600-mL beaker. The beaker was then placed in a standard refrigerator at 48C for 2 h. This protocol provided a slow cooling method that was found to cool the sample from room
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temperature to approximately 48C over 2 h. After the cooling period, the straws were loaded into a Freeze Control Cryochamber (CL-3000; CryoLogic, Mulgrave, Vic., Australia) programmed to cool from 48C to !408C at a rate of 38 min!1 (Hopkins and Herr 2010). When the unit reached its target temperature, the samples were rapidly plunged into liquid nitrogen and packed for storage.
Table 1. Brood production of instrumentally inseminated queens Individual queens from each treatment group are listed with their associated brood counts and proportion of fertilised offspring. Brood counts were taken from a single side of one frame
Thawing and motility assessment A hair dryer was fixed to a ring stand and a distance was determined at which the air coming from the unit was approximately 408C. Each straw was removed from liquid nitrogen and held at the predetermined distance for 10 s. Sperm motility was assessed for each straw before insemination. Approximately 1 mL was pipetted onto a dish and observed on an inverted microscope ("400; Eclipse TE300; Nikon, Kanagawa, Japan). A subjective motility score was assigned for each straw. Fresh semen samples contain vigorously motile spermatozoa that form swirling pockets. Motility was scored as ‘great’ if it was indiscernible from that of fresh semen. A score of ‘good’ was assigned to samples if motility was noticeably less that that of fresh semen, whereas a score of ‘poor’ was given if the spermatozoa were only slightly moving.
Initial queens (inseminated with frozen semen) Y71 Y72 Y73 Y75 Y76 First-generation queens (inseminated with frozen semen) Y53 Y54 Y55 Y58 Y59 Y64 First-generation queens (control) Y31 Y32 Y34
Instrumental insemination and experimental design Virgin honey bee queens were inseminated with 5 mL frozen– thawed or fresh (control) semen using a Harbo syringe and Schley instrument (Schley, Lich, Germany). Immediately following insemination, each queen was clipped, tagged and placed in a cylindrical queen cage in a queenless hive with young bees. Twenty-four hours later queens were treated with CO2 to stimulate egg production and placed in cages (laboratory made; 100 cm2, 0.3 cm metal wire mesh cage) pushed into a brood comb in three-frame nucleus colonies. The queens were released from the cages 72 h later and remained in these three-frame nucleus colonies throughout the experiment. The experiment began with eight sister virgin honey bee queens, each inseminated with previously frozen semen. Five of the queens were inseminated with semen frozen using Harbo’s diluent and three queens were inseminated with semen frozen with the egg yolk-free diluent. The small number of queens at the onset of the experiment and the need to produce progeny from cryopreserved semen for this experiment to continue dissuaded us from including a fresh semen control in the initial round of inseminations. Larvae needed for the production of the first-generation queens were grafted from the queen that produced the greatest majority of workers. Larvae were grafted 1 day after emergence, placed in plastic queen cup and inserted into a queenless hive with young workers. Ten of the resulting first-generation queens were inseminated with previously frozen semen using Harbo’s diluent and four were inseminated with fresh semen. Again, the queen that produced the greatest majority of workers was selected for the production of the second generation of queens. Brood evaluation Two weeks after the queen began laying eggs, a photograph was taken of the capped brood on one side of a frame
Treatment
No. capped worker cells
No. capped drone cells
% Workers
1035 52 0 20 446
0 123 106 92 0
100 29.7 0 17.9 100
240 352 0 395 75 0
177 26 114 18 179 117
57.6 93.1 0 95.6 29.5 0
1194 292 818
0 0 0
100 100 100
(43.5 " 20.64 cm). Using ImageJ64 (National Institutes of Health, Bethesda, MD, USA) and setting the scale based on the known length and width of the frame, we used the polygon selection tool to outline the brood area and recorded the area provided by the software. The ratio of workers to drones was calculated by counting the total number of capped worker and drone cells on one-half frame using ImageJ64. Brood was only assessed on the side of the frame in which the queen began laying to assess worker : drone ratios as quickly as possible so that queens could be selected for use in the production of the next generation. Results First generation Semen frozen previously using the egg yolk-free diluent appeared to be motile upon thawing, but exhibited less vigorous motility than fresh semen (recorded as good motility). However, the three queens inseminated with this semen produced only drone brood, indicating that all resulting eggs were unfertilised. Two of the five queens that were inseminated with semen that had been frozen previously with Harbo’s diluent produced a majority of workers. The other three queens produced either a mixture of workers and drones or only drone brood (Table 1). Thirty larvae were grafted from Y71; of those, 18 first-generation queens emerged. Two weeks after insemination, the mean # s.d. brood area of tracked half-frames in colonies headed by the initial queens inseminated with semen frozen with Harbo’s diluent was 320 # 41 cm2.
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Second generation (grafted queens back-crossed to previously frozen semen) Three of the four first-generation queens that received fresh semen (control) survived long enough to lay eggs. All three queens in the control group produced a majority of workers and produced an average brood area in the tracked half frame of 405.9 # 61.4 cm2. Six of the 10 first-generation queens inseminated with previously frozen semen survived long enough to lay eggs. Of those six queens, three produced a majority of workers (Table 1). Of the 30 larvae grafted from Y58, 14 second-generation queens were produced. The average brood area of tracked half-frames in colonies headed by the firstgeneration queens that were inseminated with frozen semen was 276.4 # 40.6 cm2. Although not studied directly, we observed that queens inseminated with fresh semen continued to produce fertilised offspring throughout the season (data not shown), whereas queens inseminated with frozen semen were absent after approximately 2 months. Discussion This is the first report in which two generations of queens were produced from the same stock of frozen semen. The first round of inseminations took place in early July and virgins grafted from those queens were inseminated near the end of August. The progeny of the first generation of queens were not ready for inseminations until October; thus, seasonal conditions prevented us from producing a third generation. In our experiment, we waited approximately 6 weeks between generations to check the capped brood and ascertain that we grafted only from queens producing worker (diploid) brood. Using a more typical 30-day generation time, it should be possible to generate six rounds of grafting and inseminations from a source of frozen semen, even in our northern location. Following a six-generation model, the resulting queens at the end of a single field season would have genetic compositions that were 98.4% derived from the initial source of frozen germplasm. As in any breeding programme, careful consideration must be given during the collection of semen and labelling of straws so that it is possible to avoid unnecessary inbreeding. Honey bee queens are naturally promiscuous and the collection of semen allows for the storage of semen from hundreds of males in a single straw, but it is possible to collect, label and store semen from individual hives from which a majority of the males will have been from a single mother. The preliminary experimentation with an egg yolk-free diluent was an attempt to take advantage of the benefits of egg yolk using a more defined solution. Researchers in the field of cryopreservation have been attempting to replace egg yolk for decades. Egg yolk is an animal product, the composition of which is not uniform and poses a risk of contaminating the semen (Bergeron and Manjunath 2006). Furthermore, metabolism of the egg yolk inside the queens’ reproductive tract has not been investigated and poses a risk to fertility either by clogging the sperm duct or promoting infection. The post-thaw motility of semen frozen using egg yolk-free diluent was good, although the fertilisation rate was poor. The poor fertilisation rate in the egg yolk-free diluent emphasises the importance of egg yolk as a
component of the cryopreservation diluent. The benefits of egg yolk are thought to be derived from the low-density lipoproteins present in the egg yolk (Bergeron and Manjunath 2006). Motility is crucial for fertilisation and the absence of motility would be a clear indication of failure to properly preserve the spermatozoa. However, as indicated here, the presence of motility cannot be used reliably to assess the success of the cryopreservation method. The production of fertilised eggs by queens that were inseminated with previously frozen semen is the only reliable gauge of the success of preserving the spermatozoa. Cryopreservation can be used as a method for ex situ conservation and to avoid limitations imposed by breeding seasons or weather. The methods for cryopreservation of semen in domesticated species such as cattle, horses, sheep and pigs are highly refined and it is generally accepted that one of the costs of cryoinjury caused by the cryopreservation procedure is reduced survival of spermatozoa inside the female reproductive tract (Salamon and Maxwell 1995). For that reason, although not directly studied here, cryopreserved spermatozoa in honey bee queens may not remain viable inside the spermatheca for the same length of time as fresh semen. However, for the purposes of dissemination of desirable germplasm in honey bee breeding programmes or in long-term storage within a repository, the thawed spermatozoa only need remain viable long enough to produce fertilised offspring for the production of daughter queens. When using cryopreserved semen in honey bee queens, it may be necessary to produce drones for fresh semen collection and II or to use isolated mating yards for natural mating at the end of the breeding cycle. Further investigations addressing the stability over time of the preserved sperm samples would be useful. However, the viability of properly preserved biological samples is effectively independent of storage duration. The half-life for the viability of cryopreserved material has been estimated to be .3000 years (Day and Stacey 2008). Bovine semen that had been frozen for 37 years was observed to have normal motility when thawed and was used successfully for IVF (Leibo et al. 1994). Using the methodology described in the present study, queens inseminated with previously frozen semen may reliably produce progeny. The level of viability we demonstrated in the present study is adequate to generate a substantial number of functional daughter queens (gynes) needed for dissemination of genetic stocks that are being held under cryopreservation in a genetic repository. Acknowledgement The authors extend special thanks to Susan Cobey (Department of Entomology, Washington State University, Pullman, WA, USA) for her assistance in the instrumental insemination of the virgin queens.
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