3.1. Eversion of the endophallus
Semen is collected directly from mature drones, 14 days post-emergence or older. For identification purposes, drones can be collected immediately after emergence (i.e. capturing “fuzzy” drones that are newly eclosed) and stored in cages placed in a bank colony (another honey bee colony that will tend the drones; see Büchler et al., 2013 for a discussion of “bank” colonies). Mature drones can be captured the day prior to or the day of insemination by capturing drones returning from failed mating flights or collecting them from the outside combs within the colony. To expose semen, the endophallus is readily everted by hand in two-steps: the partial eversion, and the full eversion.
Maintain sanitary conditions, as drones often defecate during the procedure. Hold the drone to avoid the endophallus touching the drone body or your fingers and keep a towel soaked in alcohol to clean up. The eversion of the endophallus is preformed within a few seconds. Evaluation of drone maturity and semen quality must be determined instantly; any drone that does not evert properly or does not present sufficient (~1 µl) semen on the bulb (see section 3.2) should be discarded. Semen collection is tedious, therefore proper techniques and practice will greatly increase efficiency. Plan to have a plentiful supply of mature drones, more than is needed, as not all will yield semen. Keep drones warm and well fed until they are used. A light above the flight box provides warmth and bee candy or a piece of honey comb will extend their activity.
Procedure for everting drones:
- Assemble and prepare the syringe
a. All parts should be sterilized, either by heat or an alcohol wash, and rinsed with distilled water.
- To obtain partial eversion, grasp the head and thorax of the drone between the thumb and forefinger, ventro-dorsally, with the abdomen facing upward. It is helpful, if the individual is right-handed, to hold the drone’s head with the right hand and squeeze the abdomen with the left so that the drone remains held in the left in position for sperm collection.
- Roll or crush the thorax between your fingers.
a. If mature, the abdomen will contract and a pair of yellow-orange cornua emerge (Fig. 5).
b. If the abdomen remains soft or the cornua lacks colour, the drone is immature and will not yield semen (Fig. 6).
- To obtain full eversion, grasp the base of the abdomen near the thorax
with the thumb and forefinger and apply pressure along the sides of the
abdomen, starting at the anterior base and working toward the posterior tip.
a. Squeeze and roll your fingers together in one steady forward motion, forcing the eversion to complete.
b. Hold the drone with his abdomen pointing downward to keep the endophallus from falling back onto your fingers and contaminating the semen as in Fig. 7. This positioning also provides ready placement under the microscope.
c. The exposed semen is a creamy, marbled tan colour, with an underlying layer of white mucus (Fig. 8).
Fig. 5. Partial eversion of the endophallus of a mature drone at early (left) and late (right) partial eversion. At this stage, the abdomen will contract and a pair of yellow-orange cornua appear.
Fig. 6. Partial eversion of the endophallus of an immature drone. The abdomen is soft and the cornua lacks colour.
Fig. 7. Semen contaminated. Avoid contamination of semen during the eversion process. Position the drone to stop the endophallus from falling back onto your fingers or the drone’s abdomen.
Fig. 8. Eversion of the endophallus with semen exposed. The exposed semen is a creamy, marbled tan colour, with an underlying layer of white mucus.