Improving Strains of Bees by Artificial Insemination

New Zealand Journal of Agriculture, Volume 81, Issue 2, 15 August 1950, Page 101

Improving Strains of Bees by Artificial Insemination

'T’HE application of artificial insemination of queen bees at the Department of Agriculture’s Animal Research Station, Wallaceville, to a project for the improvement of strains of bees in New

Zealand is described in this article. by

T. Palmer-Jones,

a Research

Officer at the station, and

G. E. Miller,

Horticultural Cadet, Auckland.

Artificial insemination is the only method showing promise of being of practical value in the removal of the element of chance from the mating of queen bees and its replacement by. a controlled method. Application of the method is still in the experimental stage, but great possibilities of improving strains of bees are evident.

COMMERCIAL beekeepers have long realised that the progeny of queen bees vary a great deal in their capacity to gather honey, tractability, swarming tendency, and other qualities. Selective breeding of queen bees for desirable qualities is the accepted practice, but in the past it has been much handicapped by the fact that the queen bee mates only in the air and not necessarily with drones from her own hive. Commercial breeders of queen bees usually attempt to overcome this difficulty by placing their queen-rearing apiaries in isolated localities and stocking them entirely with what they regard as suitable bees for breeding. Under these conditions the air surrounding the apiary is saturated with drones of the right type and mismating is unlikely to occur. At best this method gives somewhat haphazard results and does not permit improvement of strains of bees by the methods which have been used with success in the breeding of domestic animals. Many unsuccessful attempts were made to remove the element of chance from the mating of queen bees and to replace this with a controlled method. These attempts included mating the queen with approved drones in enclosures, mating the queen with a drone by the manual manipulation of the

operator, and early attempts at artificial insemination. Artificial insemination was the only method which showed promise of being of practical value. After much painstaking work, Watson in America was able to demonstrate in 1926 a satisfactory method of artificially inseminating queen bees. Since then many American workers have gradually improved the instruments used, and the technique of insemination. The apparatus now used for artificial insemination, the technique of the method, and sufficient of the anatomy of the reproductive organs of bees for its understanding are described in a manual by Mackensen and Roberts (1) from which the following account is largely taken. Anatomy of Queen Bee In brief, instrumental insemination consists of removing semen from selected drones with a microsyringe and injecting it into the oviducts of a virgin queen bee. To make the process clear it is necessary to describe the anatomy of the queen bee in some detail.

1. Mackensen, 0., and Roberts, W. C. (1948): “A Manual for the Artificial Insemination of Queen Bees,” United States Department of Agriculture.

The tip of the abdomen of the queen consists of an upper, or dorsal, plate and a lower, or ventral, plate, which close at the tip like the two halves- of a shell. The cavity enclosed by these plates is called the sting chamber. In Fig. 1 the tip of the abdomen is shown in proper position for artificial insemination, with the dorsal plate and the ventral plate drawn apart to expose the sting chamber and its structures, including the sting and the vaginal orifice.

HEADING PHOTOGRAPH: The apiary used in the insemination project at the Animal Research Station, Wallaceville. -

Fig. 2 illustrates the internal portions of the reproductive tract with the side toward the observer removed. A fold across the anterior floor of the sting chamber loosely separates a region called the bursa copulatrix from the sting chamber proper. The vagina, through its vaginal orifice, and the bursal pouches open into this region. The spermathecal duct from the spermatheca enters the vagina in front from above. Just below the opening of this duct is the valvefold, a large, tongue-like structure with transverse ridges, which make it distinguishable from other tissues when viewed through the vaginal opening. Its position is such that it can close the passage between the vagina and the middle oviduct with a valve-like action. The paired oviducts enter the middle oviduct anteriorly. They are large fluted structures capable of great expansion for the temporary storage of sperm after mating and of eggs in a laying queen. Each paired oviduct leads to an ovary. In Fig. 2 the reproductive tract is extended, but during insemination the queen is held in such a way that the vagina is collapsed, and the valvefold often appears

to be just inside the vaginal opening (Fig. 1). A knowledge of the anatomy of the reproductive organs of the drone is not required for artificial insemination. In order that the semen may reach its natural position by artificial insemination, the valvefold must be pushed down to permit the point of the syringe to pass into the middle oviduct. If the syringe enters only the mouth of the vagina, the semen presses the valvefold against the middle oviduct and is forced back around the syringe and out into the sting chamber. The vagina is-not easily distendable, but the oviducts expand to hold a large quantity of semen. Equipment Used A microscope and microscope lamp, an apparatus for delivering a steady flow of carbon dioxide, and a manipulating apparatus for holding the queen and instruments during insemination constitute the equipment used. Microscope: The collection of semen from selected drones, the preparation of the queen for insemination by open-

ARTIFICIAL INSEMINATION OF BEES

ing the sting chamber with instruments, and the insemination itself are all carried out under a microscope with a strong light focused on the queen. A magnification of 22.5 times is commonly used in the work. In Fig. 3 the operator on the right is shown using the apparatus to inseminate a queen while the operator on the left uses similar apparatus to load a syringe with semen from a drone. Carbon dioxide supply: A very slow stream of carbon dioxide gas is passed over the queen to keep her anaesthetised while she is held in a plastic queen holder for insemination. The gas is supplied from a cylinder in which it is confined at a high pressure, and the pressure must be reduced before the gas reaches the queen. The apparatus for this purpose is shown in Fig. 4. At Wallaceville the ordinary diaphragm reducing valve (B), though capable of reducing the gas pressure to a few pounds, was found to be too insensitive. The difficulty was overcome by passing the gas, after it left

ARTIFICIAL INSEMINATION OF QUEEN BEES

the reducing valve, into a small chamber (C) fitted with leads the entry of gas into which could be controlled by fine needle valves. From one of these leads the gas was passed, through a bubbler tube (D) partly filled with water to the plastic tube (E) which delivered it to the queen holder. The gas could also be passed directly from the reducing valve (B) through a tube which could be closed with a stopcock and through the bubbling system (F) to the container (G) which' was used for anaesthetising caged queens. _ The supply of carbon dioxide to the queen could be gauged easily by the rate of bubbling in the bubbler and regulated to give the minimum flow needed to keep the queen completely anaesthetised during insemination. The manipulating apparatus consists essentially of a heavy stand on which are mounted the queen holder, the syringe, and the holding hooks in such a way that they can be moved and adjusted easily (Fig. 6). Syringe and instruments: A and B in Fig. 7 are the components of one of the latestpattern syringes. It consists of a metal plunger which can be screwed up and down a

barrel to exert pressure on a rubber diaphragm by way of an intervening piece of pointed metal. A plastic screw tip holds the diaphragm in place. Before use the

syringe tip is unscrewed and its base filled with water. Then the semen is sucked into the tip by screwing the plunger back in the barrel. The probe (D) is used to push down the valvefold so that the syringe can be inserted in the vagina. The holding hooks (E and F) are used to hold the sting chamber of the queen open during insemination. E is the ventral hook, which fits over the ventral plate, and F the dorsal or sting hook used to press the sting out of the way. A blunt dissecting needle is used to push down the sting so that the sting hook can be placed in position. The manipulating apparatus, syringe, and instruments described were bought from the Department of Economic Entomology, University of Wisconsin, U.S.A. In the apparatus supplied the syringe mounting block could be adjusted to no greater extent than the ventral-hook and sting-hook mounting blocks. It was found of great convenience, however, to modify this part of the apparatus slightly by passing the screw supporting the syringe holder through a horizontal slot cut in a plastic syringe mounting block, which was then mounted on the metal

pillar. The screw was flattened and fitted the slot closely so that it could not turn. ' The syringe holder could thus be moved in the horizontal plane in the same fashion as the queen holder. This arrangement allowed greater flexibility in adjusting the instruments before insemination and reduced the time of the operation. The queen holder is a tube tapered at one end and with a movable stopper fitting closely inside. The queen is backed into the tube and held with the stopper so that her abdomen projects beyond the end. The stopper is bored out so that carbon dioxide can be allowed to flow round the queen, so keeping her anaesthetised during insemination. Insemination Technique The queen is run into a plastic tube of the same diameter as the queen holder. This is fitted with a plastic stopper which can be pushed along inside it and used to coax the queen tail first into the queen holder. The queen is always arranged so that when she is anaethetised her upper surface is to the right of the operator. The two holding hooks are then inserted, one at a time, into the sting chamber and the abdominal plates pulled apart, the operation being performed under the microscope. The syringe is then E laced horizontally in its older so that its tip is in focus, ready to be filled with semen.

The selected drones are anaesthetised by dropping them into a stoppered glass jar with a wad of cottonwool soaked in chloroform covering the bottom. The penis of a sexually mature drone usually everts partially under this treatment, and more complete eversion is secured by squeezing the drone between the fingers. The semen is cream coloured and can be distinguished from the white and more viscous mucous. When the penis is at the right stage of eversion for withdrawal of semen it is brought in contact with the tip of the syringe and the semen skimmed off by withdrawing the plunger (Fig. 8). Inseminations at Wallaceville were usually made with the semen from three or four drones.

Immediately after being filled the syringe is placed in position above the queen, its tip having been . moistened with water to act as a lubricant. The sting hook is pulled so that the sting chamber appears as in Fig. 1. Then the probe is inserted into the vagina and the valvefold pushed down so that the syringe can pass over it into the median oviduct (Fig. 9). When the syringe is in position (Fig. 10) the plunger is slowly screwed down sc that the semen enters the oviduct. Il semen begins to well out round where the syringe enters the vagina of the queen, it indicates that the tip of the syringe is not properly in place and the syringe must be withdrawn and reinserted. Though at first some trouble was experienced at Wallaceville in mastering the insemination technique, it was found that after several weeks of practice the operation could be performed without much difficulty. At this stage of proficiency the time required tc remove a queen from the adjoining apiary and inseminate and return he] averaged about 30 minutes. Inseminations, usually two, are made from the fourth to the tenth day aftei the queen emerges, and should be twc days apart. Conduct of Project Artificial insemination of queen bees has provided a practical means of improving strains of bees but, though t satisfactory .. technique has beer evolved, its application to the bes' advantage is still in the experimenta: stages. From work carried out ir America it appears that the only practical method is first to inbreed strains of bees and then to cross the inbrec strains to produce hybrids.

ARTIFICIAL INSEMINATION OF BEES

The Wallaceville project entails first inbreeding selected queens, then crossing the inbred strains to produce hybrids, and finally testing the worth of these hybrids and their progeny by the criteria used in judging the value of queens raised commercially. Crossing of the inbred strains to produce hybrids is necessary because inbreeding reduces fertility, which this procedure restores. Inbreeding concentrates good and bad qualities, so that, though many hybrid queens, will be much below the average in performance, others can be expected to be above the average. At this stage of the programme, progeny testing will be carried out so that the best hybrid queens can be selected. These selected queens will be placed in isolated apiaries and young queens bred naturally from them tested further in these apiaries. Queens from the best hybrid strains will then be used for production of young queens by natural mating for further testing in commercial apiaries and at Wallaceville. Finally, suitable hybrid strains will be chosen for mass production of breeder queens, by natural mating, for supply to the industry. This programme will require at least 5 years for completion. It is hoped in this way ultimately to supply the industry with queens better than those at present in use. As the desirable qualities of the selected queens become diluted by natural mating, it will be necessary to renew the strains from the laboratory apiary. An association of breeders of queen bees has been formed to provide suitable stock on which to base the project and to . furnish the necessary organisation for testing strains of queens produced in the course of the work. In October, 1949, 15 members of the association gave a total of 50 queens which are now established in nuclei in the laboratory apiary at Wallaceville and have provided all the stock at present being used in the insemination project. . Suppliers of queen bees were sent report sheets on which they noted the following particulars for each queen: Age of queen (month and year bred), longevity and stamina of strain, development and maintenance of brood nest, physical characteristics of progeny (males and females), temperament of progeny and quietness on combs when handled, swarming tendency, economy of winter stores, use of propolis, production of honey, seasonal conditions, main nectar sources worked by the colony, and general remarks. Thus a complete record of each queen bee was available for reference. Recording System Particulars of each queen were noted on numbered cards and discs bearing the same numbers were affixed to the appropriate nuclei. The number allotted the original queen remained constant for her and all queens . bred from her, but this number was followed by a small 1 in the case of the original queen, by a 2 for her daughter, by a 3 for her granddaughter, and so on. By referring to numerals on nuclei in the apiary it was possible to tell how many generations the queens were removed from the original queen.

ARTIFICIAL INSEMINATION OF BEES

The ages of the queens ranged from a few months to 2 years, the average being about a year. Judged by their performance at Wallaceville, which ranged from fair to good, these queens appeared a representative section of the breeders’ stock. * , Prevention of Natural Mating On arrival at Wallaceville the queens given by breeders were placed in nuclei with strips of hinged queen excluder fitted across the entrances (Fig. 11). Throughout the work all descendants of these queens were placed in similarly treated nuclei, so that neither drones nor queens could enter or leave. Thus virgin queens were retained, without being able to leave the hive to mate naturally, until they were ready for insemination, and all drones in a nucleus were known to have come from its queen and not to have drifted in from other hives. Type of Nucleus Hive Used Nuclei were used throughout the insemination programme because of the ease and convenience with which they allowed bees to be handled. The most satisfactory type for much of the work was found to be a double, nucleus formed by dividing a standard hive body with a division board and placing this on a flat bottom board. A hive mat was nailed down to the division board so that when bees were being handled on one side of the division board the mat could be folded back over those on the other side. Each nucleus had its own entrance hole and a ventilation hole covered with wire gauze, which were at opposite ends of the double nucleus above small projecting alighting boards. Such an arrangement allowed first and second generation queens to be established side by side, which was very convenient in apiary manipulation. Ordinary single nuclei were also used (Fig. 11). Provision of Drone Comb Large supplies of drone comb were required for insertion in nuclei so that drones could be raised from queens suitable for insemination. This comb

was provided by inserting frames with small “starters” of foundation comb in strong hives, which were fed sugar syrup when the honey flow was insufficient to stimulate wax production. Returning- Queens after Insemination Virgin queens were located as soon as possible after being hatched out and replaced in their, nuclei inside queen cages closed with a removable cork plug. These queen cages were provided with small metal strips fastened at right angles to the tops so that the cages could hang between the frames and allow the bees easy access to the queens through the gauze. Queens were kept in the cages throughout the period during which they were being inseminated. Two days after the final insemination the cork plug • was replaced with a plug of candy through which the bees could eat their way to liberate the queen. The use of this method reduced losses of queens caused by balling and maltreatment by the bees. ’ When she was removed for her first insemination each queen was marked with a white spot on the thorax and one of her wings was clipped (Fig. 12). This procedure ensured that subsequently queens could be found and observed easily. Two Methods of Inbreeding During the 1949-50 season inbreeding of queens at Wallace was carried out by two methodsbrothersister mating and mother-son mating. Brother-sister Mating The first system depends on mating a virgin queen with her brothers. A queen is removed from a nucleus and the virgin queen raised by the . bees from one of her eggs inseminated with semen from her mother’s drones. When this artificially inseminated queen is laying she is removed and the procedure repeated for two more generations, except that drones from the same source are used (back crossing). Then true brother-sister mating is used again. This is a slower method of inbreeding than the mother-son system, but it is much easier to carry out.

ARTIFICIAL INSEMINATION OF BEES

Mother-son Mating It has been found that egg laying of virgin as well as inseminated queens can be stimulated by treatment with carbon dioxide. This causes the queens to begin laying almost as soon as naturally mated ones, whereas without it only about one in five starts earlier than 30 days after emergence. Two carbon dioxide anaesthetisations of 10 minutes, spaced a day apart, are used to start egg laying. The second treatment of the queen must be completed before the sixth day after emergence if she is to begin laying at the normal age (8 to 11 days after emergence). Thus drones can be obtained from virgin queens and later the virgin Queens can be self-fertilised by inseminating them with semen from their own drones (2). According to Kalmus and Smith (3), mother-son mating is much the most efficient method of inbreeding bees to. secure pure lines. Comparison of Methods The tables which follow compare the times required to complete one cycle of inbreeding with each method. The figures quoted are approximations. Brother-sister Mating In this method the nucleus must contain drone brood 14 days old before the queen is removed for the first inbreeding, so that the drones, which develop more slowly than queens, will be ready in time to provide semen for inseminating the virgin queen. Time required for development of drone brood .. 14 days Time required for bees to raise a virgin queen . . 10 days Time between emergence of queen and completion of second insemination . . 7 days Time required for queen to begin laying . . . . 6 days Total time .. . . . . 37 days

2. Mackensen, O. (1947): “Journal of Economic Entomology” (U.S.A.), volume 40, number 3, pages 344-349. 3. Kalmus, H., and Smith, C. A. B. (1948): “Journal of Genetics” (Cambridge, England), volume 49, part 2, pages 153-158.

In subsequent inbreedings the total time required would be either 23 or 37 days, depending on whether drones from the original hive (back crossing) or brother drones were used. Mother-son Mating In this method the queen is first removed from a nucleus. Time required for bees in nucleus to raise a virgin queen 10 days Time between emergence of virgin queen and completion of second carbon dioxide treatment . . 6 days Time required for queen treated with carbon dioxide to begin laying in drone comb . . . . 10 days Time required for drones to reach sexual maturity .. 30 days Time required for queen inseminated with these drones to start laying . . 4 days Total time . . . . . . 60 days According to Kalmus and Smith (3), a complete cycle of mother-son mating (a double generation) . is more than three times as effective as a complete cycle of the brother-sister method of inbreeding. However,. 60 days are needed to complete a cycle of the first method, compared with 37 or 23 days for the second, so that if both were applied for the same period the mother-son method would be about one and a half times as effective for inbreeding as the other. The motherson method, however, is . the more difficult to apply. . • The mother-son method depends on the bees tolerating a drone-laying queen for the period of some 30 days necessary for her eggs to develop into sexually mature drones which can provide semen for her insemination. It has been found that such a queen frequently is balled and finally killed by the bees in an attempt at supersedure. The fates of 23 queens inbred by the mother-son method at Wallaceville during the 1949-50 season were: —

Five queens accepted after the carbon dioxide treatment could not be inseminated, as their drones were not sufficiently mature. Undoubtedly the losses shown were caused by balling and maltreatment of the queens by the bees, as these were noticed on many occasions when the nuclei were inspected. . When balling of a queen was noticed the queen was smeared with honey and returned to the nucleus in a hanging queen cage closed with candy. Usually the method •helps toward the acceptance of a queen by bees, but in this case it proved of little value, as did all other, methods tried. It was soon apparent that the mother-son method of inbreeding was not practicable, and it was abandoned and the brother-sister method alone employed. The impractability of the motherson system of inbreeding was confirmed by Dr. W. C. Roberts, of U.S.A., in a recent letter. He states that the degree of insemination of such queens is usually very low and frequently they become drone layers when overwintered. However, the brother-sister method allows 100 per cent, insemination and the queens can be overwintered satisfactorily. Scope of Project Forty-one . queens were inbred by the brother-sister method at Wallaceville during the 1949-50 season. Thirtyeight of these were inbred for one generation and three were inbred for two generations. Twenty queens of those inbred for one generation were not accepted by the bees and were found dead after preliminary maltreatment. One queen of those inbred for two generations was lost. The extent to which inbreeding will be carried out will be governed to some degree by the ■ hatchability of eggs of the inbred queen, but will extend for several generations wherever possible. Throughout the work it has been found that the frequent handling of queens, the constant opening and closing of nuclei, and the use of queen excluders to bar the entry and exit of drones and queens all create highly artificial conditions which make the bees much less willing to accept queens than they would be if the queens had been allowed to mate naturally. Though artificial insemination of queen bees offers great possibilities of improving strains of bees in New Zealand, its application is still in the experimental stage and a long period must elapse before the beekeeping industry can benefit from the work initiated at Wallaceville.

Total number of queens treated with carbon dioxide: 23 Total number of queens accepted after carbon dioxide treatment: 11 Number found dead shortly after treatment: 12 Number accepted by bees: 11 Number of accepted queens inseminated with semen from own drones: 5 Number of inseminated queens accepted by bees: 1