Apicultural Research

A New Mini Syringe for Multiple Inseminations of Honey Bee Queens

by TIAGO MAURCIO FRANCOY1 and LIONEL SEGUI GONALVES2 Manuscript received for publication June 3, 2004
ABSTRACT
Instrumental insemination of queen honey bees has been very useful for genetics studies, maintenance of bee lines and genetic improvement programs. We developed a new, inexpensive mini-syringe device, capable of multiple inseminations. The syringe itself is a plastic insulin syringe, and the base and holder are made of stainless steel. The syringe tip consists of a glass capillary tube, which contains the semen, attached with silicone tubing. This insemination device is simple and inexpensive to build and maintain, and it is easy to manipulate. We initially tested the mini syringe by inseminating 12 queens; all of them survived and started to lay eggs, which originated worker larvae within a few days after the inseminations. The main advantages of this new syringe model (the Francoy-Gonalves Mini Syringe) are the possibility of multiple inseminations, which saves time in the insemination process, and the fact that it is constructed with inexpensive, but durable material. The entire process became faster, easier and more efficient. The new mini syringe also allows us to inseminate queens with stored semen in glass capillaries, by just changing the insemination tip for another pointed capillary with stored semen.

Mackensen, 1948, 1954; Mackensen & Roberts, 1948; Laidlaw, 1949a,b). The main contribution of Mackensen was the development of the syringe that is now part of all models of inseminators. The model developed in 1948 incorporated a liquid plunger in a removable plastic tip. The liquid plunger was controlled by a diaphragm made of rubber, set in the base of the tip. This syringe is still used widely, and it allows full insemination of a single queen or a couple of queens, depending on the amount of semen in the tip. Professor Warwick E. Kerr first introduced instrumental insemination into Brazil, using the equipment developed by Laidlaw, which had the Mackensen syringe adapted to the inseminator. Later, Gonalves & Brites (1970), while working in Kerr's lab, developed a new model of instrument whose design was based totally on screws, avoiding the utilization of "racks and pinions", therefore of easier construction. The syringe in this instrument was based on Mackensen's design.

INTRODUCTION ince its development, the instrumental insemination of honey bee queens has been used mainly for research. This technique has made possible total control of mating, the maintenance of honey bee lines and genetic markers, and the technification of selection and genetic improvement programs. Since instrumental insemination techniques were first developed, various changes have been made. The first attempt to instrumentally inseminate queens was made by Francis Huber, who painted drone semen on the vaginal orifice of queens with a brush (Huber, 1814). However, he did not succeed. Many other attempts have been made by various researchers, but without success. It was only in 1927 when L.R. Watson published his research that the technique of instrumental insemination of queens was accepted as viable. Later, one of his students, Nolan (1932, 1937) improved the technique and instruments, and these were used as a basis for the model developed by Roberts & Mackensen, H.H. Laidlaw and others, whose instruments are widely in use today (Roberts, 1947;

Figure 1: Lateral view of the Francoy-Gonalves Mini Syringe.

1 - Genetics Department, Medical School, University of So Paulo, 14.049-900 Ribeiro Preto, SP, BRAZIL tfrancoy@rge.fmrp.usp.br 2 - Biology Department, Faculty of Philosophy, Sciences and Letters, University of So Paulo, 14.040-901 Ribeiro Preto, SP, BRAZIL lsgoncal@usp.br Telephone number: +55 (16) 602 3154 Fax number: +55 (16) 633 6482 Corresponding author e-mail: lsgoncal@usp.br

Figure 2: Close-up view of the Francoy-Gonalves Mini Syringe.

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In 1976, Gonalves reduced the length of the Mackensen syringe to facilitate its use in Laidlaw's apparatus. An important improvement of the instrumental insemination syringe is the Harbo syringe, which uses glass capillary tubes and allows multiple inseminations of queen bees without the need for individual semen collection for each new queen (Harbo, 1985). We have developed a new mini syringe, also capable of multiple inseminations. This syringe is both simple and inexpensive to build and maintain, and easy to manipulate (Figures 1 to 5).

The development of the Francoy-Gonalves Mini Syringe was based on the design and working principles of the Harbo syringe. It consists of an exchangeable plastic cylinder (a 1 mL insulin syringe), linked to a silicone capillary tube that is graduated in 1 1 (Figure 3). There is also a glass capillary with a tip that works as a syringe tip for the insemination (Figures 4-5). The other parts of the apparatus are a plunger and the metal support of the syringe (Figures 1-2). The metal support is made of stainless steel. It is constituted of a circular base linked to a cylindrical column that supports the

Figure 3: Technical drawing of the mini syringe.

Figure 4: Insemination of a queen honey bee with the mini syringe.

Figure 5: Close-up of insemination with the mini syringe.

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mini-syringe body (Figure 3). The mini-syringe body consists of a metallic cylinder, where the screw plunger is located; the exchangeable plastic cylinder is on the opposite end. The plunger is directly linked to a screw, which allows for accurate control of plunger displacement. The capillary tip is fixed to a 4 cm long, modified Pasteur pipette that works as a syringe tip socket, to which the syringe tip is tightly fixed. This modified pipette is used as a support in the Gonalves syringe holder (Gonalves and Brites 1970) and in the Laidlaw instruments, since the glass capillary tube is too thin to be used without this complement. The main advantages of the Francoy-Gonalves insemination device are the possibility of multiple inseminations, which saves time in the insemination process, and the fact that it is constructed with inexpensive, but durable material. The most time-consuming procedure during the normal process of insemination is the collection of semen from the drones. This new insemination device allows semen collection from many drones in succession, avoiding the repetition of the entire process for every new queen. Our tests showed that the insemination of three or more queens became easier and faster than with traditional equipment, using the Mackensen syringe model or the model developed by Gonalves. Another advantage is the facility of sting chamber visualization during the introduction of the tip into the queen vaginal orifice. The glass tip has a smaller diameter than the plastic tip, which makes the visualization of the vaginal valve fold and the introduction of the capillary tube tip behind the valve fold easier. Currently there are several ways to store semen; storage in glass micro-capillary tubes is quite common. Thus, it is also possible to inseminate queen bees with semen stored in glass capillaries by just changing the insemination tip for a capillary with stored semen and using it as a syringe tip. We tested the Francoy-Gonalves Mini Syringe on 12 queens. All of them survived and soon laid eggs, which originated worker larvae a few days after the insemination. ACKNOWLEDGMENTS The authors thank Marcela B.F. Laure and Joo Jos dos Santos for technical assistance with insemination and in the apiary and Luis Augusto Magrini for the construction of the mini syringe. David De Jong helped correct the language.
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LITERATURE CITED Gonalves, L.S. (1976). Seleo direcional em duas linhagens endocruzadas de Apis mellifera L. (Directional selection in two inbred lines of Apis mellifera L.) Associate Professor Thesis. Genetics Department, University of So Paulo, Ribeiro Preto. Gonalves, L.S. & Brites, J.P. (1970). Nota sobre um novo instrumental usado na inseminao de rainhas de himenopteros. (A note on a new equipment used for the insemination of hymenoptera queens). Proceeding of the I Brazilian Apiculture Congress, Florianpolis-SC. pp. 119122. Harbo, J.R. (1985). Instrumental Insemination of queen bees 1. Amer. Bee J. 125: 197-202. Huber, F. (1814). New observations upon bees. C.P. Dadant. Amer. Bee J., Hamilton Ill. Laidlaw, H.H. Jr. (1949a). New instruments for artificial insemination. Amer. Bee J. 89: 566-567. Laidlaw, H.H. Jr. (1949b). Development of precision instruments for artificial insemination of queen bees. J. Econ. Ent. 42:254-261. Mackensen, O. (1948). A new syringe for the artificial insemination of queen bees. Amer. Bee J. 88:412. Mackensen, O. (1954). Some improvements in method and syringe design in artificial insemination of queen bees. J. Econ. Ent. 47:765-768. Mackensen, O. & W.C. Roberts. (1948). A manual for the artificial insemination of queen honey bees. U.S.Bur.Ent.and Plant Quar.Et-250. Nolan, W.J. (1932). Multiple matings of the queen bee. In: Maryland State Bee Keepers Association Report, 23rd. Annual Meeting pp 20-34. Nolan, W.J. (1937). Improved apparatus for inseminating queen bees by the Watson method. J. Econ. Ent. 39:700705. Roberts, W.C. (1947). A syringe for artificial insemination of honeybees. J. Econ. Ent. 40: 445-446. Watson , L.R. (1927). Controlled mating in the honey bee. Rept. State Apiarist, Iowa, pp 36-41.

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