Preparing The Queen

The queen is made to walk into a tube similar to the queen holder. When she reaches the constricted end she backs up, and if the queen holder is quickly put in place, she usually backs in readily. As soon as she reaches the constricted end of the queen holder, the stopper through which carbon dioxide is flowing is quickly pushed in after her (before she has a chance to move forward again). To be properly positioned the last three visible segments of the abdomen should protrude, and the hind legs should remain in the queen holder. A beginner may wish to insert a 3-way stopcock in the carbon dioxide supply tube so the gas can be diverted while the queen is placed. (If the queen's abdomen expands abnormally, the gas is being forced into her abdominal air sacs, an indication that the flow is too strong. ) The queen holder is then put in place on the manipulating apparatus and adjusted so that the dorsal surface of the queen's body is to the right.

As soon as the queen is quiet, the sting chamber can be opened with the holding hooks, with the ventral hook to the left and sting hook to the right. The hooks are manipulated more or less simultaneously, but usually first the ventral hook and then the sting hook are inserted into the sting chamber and abdominal plates pulled apart. With the left hand, the sting depressor is used to hold the sting down while the sting hook is placed in the triangular area between the bases of the sting lancets, and left in this position to prevent unnecessary drying of the delicate tissues while the syringe is being loaded.

Ejaculation Of Drones

Drones vary greatly in the ease with which they are induced to ejaculate and in the amount of semen delivered, especially when one is dealing with inbred drones. Some have no semen; others may not evert and ejaculate properly when the stimulation is artificial; still others may evert so violently that the semen is projected and lost or the penis explodes.

The method commonly used to induce eversion and ejaculation is the following: The drone is decapitated and, if this does not cause a partial or complete eversion, he is grasped by the thorax with the thumb and forefinger of the left hand with the drone's ventral surface up. Then, while the anterior part of the thorax is squeezed with the left hand, the dorsal part of the abdomen is teased or repeatedly squeezed lightly with the thumb and forefinger of the right hand. This action usually brings about contraction of the abdominal muscles and a partial or sometimes a more or less complete eversion and ejaculation (fig. 4). If eversion is only partial (fig. 4, A), the abdomen is squeezed progressively from the anterior dorsal region to the posterior ventral part to continue the eversion by force until the semen is extruded (fig. 4, B or 4, C). Semen is rarely obtainable without abdominal contraction, but when the abdomen contracts without partial eversion, the eversion can often be completed by pressure and a good amount of semen obtained.

The amount of semen and mucus varies. The cream-colored semen comes out first followed by the thicker white mucus. Sometimes only semen is ejected, but usually at least some mucus comes out after the semen, and the two are distributed on the penis in various arrangements. Movement of sperm causes the semen to spread in a thin layer over the mucus, making it progressively more difficult to collect. Also, delay after the first stage of eversion makes the semen more difficult to collect, possibly due to a mixing of semen and mucus. It is, therefore, important to complete the whole procedure as quickly as possible.

Sometimes drones evert and ejaculate better if they are first induced to exercise by holding them by the legs with forceps while they attempt to fly or by permitting them to fly against a windowpane. Sometimes caging free-flying drones for a day or two improves eversion and ejaculation. Perhaps these methods increase the volume inside the abdomen by forcing air into the air sacs or by increasing the accumulation of feces; thus the contraction of the abdominal muscles is more effective in everting the penis.

Sometimes exposure to chloroform fumes is effective when all other methods fail; in fact, it is quite reliable in causing at least a partial eversion; however, an experienced operator will usually find this method too time-consuming.

Filling The Syringe

This operation is done under the microscope with the syringe tip swung a few inches toward the operator and the microscope moved correspondingly. The ejaculated drone is brought near the syringe tip with the left hand. The plunger of the syringe is withdrawn slightly to provide an air space (as small as practical-one-half microliter) between saline solution and semen and to facilitate measuring the amount of semen. The surface of the semen is then made to touch the point of the syringe at about a 45° angle. If the drone is pulled away from the syringe slightly without breaking contact, the semen will continue to adhere to the syringe and will flow toward it as the plunger is withdrawn. This procedure helps the operator to avoid taking up the mucus which is too thick to pass into the syringe tip and will stop the passage of semen. If mucus clogs the tip, the plunger is pushed out until the passage is cleared; then the taking of semen is resumed. By moving the drone about in relation to the syringe tip, the mucus can be skimmed of practically all its semen covering. Semen is taken from as many drones as necessary, and when the syringe tip is filled to the desired point a small amount of air or physiological salt solution is drawn in to prevent sealing of the end of the syringe tip by drying of semen. This air or salt solution can be injected with the semen without harm. The average yield of semen per drone is about 1 microliter. The beginner should take up semen very slowly; this is very important. Speed will come with experience.