^ARY February 194S ET-250 United States Department of Agriculture Agricultureil Research Administration Bureau of Entomology euad Plant Quarantine A MANUAL FOR THE ARTIFICIAL INSEMINATION OF QUEEN BEES By Otto Mackensen and W. C. Roberts Division of Bee Culture -2- CONTENTS Page Introduction • • • 3 The reproductive organs ..... 3 Bquipnent and oonstruotion of instruments ...•••..... 5 Miorosoope and light •• ....... 5 Carbon dioxide equipment ••••« •• 5 The manipulating appeu-atus 6 Queen holder 6 Syringe •• 7 Glass syringe .* • 8 Plastic syringe • 10 Holding hooks «.•••• . . 11 Other equipment • 12 Insemination procedure • 12 Adjusting the flow of carbon dioxide •........• 12 Preparing the syringe •..••• • 12 Preparing the queen 13 Filling the syringe IS Injection 15 Cleaning the syringe • 15 Sterilizing the syringe • • 16 Estimating the number of sperms 16 The use of carbon dioxide 17 Procedures for Tarious types of matings and results to be expected •• • 18 Individual matings • 18 One insemination with sperm from several drones 19 Two or more inseminations 19 The absolute control of parentage 20 Care of queens ••• • 21 Bearing and care of drones 22 Summary • •••.••• 24 Literature cited 26 -3- A UAUUkL FOR THE ARTIi-'IClAL INSEMINATION OF QUEEN BEES By Otto Mackensen and W. C. Roberts,^ Division of Bee Culture INTRODUCTION Ever since the discovery that the queen mates in the air, the con- trol of mating has been the bee breeder* s dream* A number of methods have been devised for oontrolling the natural mating act, of which iso- lation at mating stations has been the most satisfactory. It has long been realised, however, that absolute control of mating could only be attained through some method of artificial insemination, and many efforts have been made in this direction with varying degrees of success. The many early attempts at artificial insemination have been reviewed by Nolan (^) and, although the early investigators have advanced the method, the works which have contributed most are those of Watson (£, 10 ), Nolan (_5, 6) , and Laidlaw (2_) • By modifying the apparatus and methods devised by these three men, the writers have been able to obtain results far superior to any heretofore presented, and it is the purpose of this publication to describe the apparatus and procedure in sufficient detail so that anyone may duplicate them.2/ THE REPRODUCTIVE ORGANS To be successful with artificial insemination one must be familiar with certain anatomical features of the queen. Although several workers have studied the sex organs of bees, Laidlaw (2^) made the most recent and thorough study from an artificial-insemination standpoint, and was the first to recognize the full significance of the valvefold of the queen in the success of the insemination process. The tip of the abdomen of the queen is made up of an upper, or dorsal, plate and a lower, or ventral, plate, which close at the tip like a clam shell. The cavity that these plates enclose is called the sting chamber. In figure 1 the tip of the abdomen is shown in proper position for artificial insemination, with the dorsal plate (DP) and the ventral plate (VP) drawn apart exposing the sting chamber and its various structures, including the sting (ST) and the vaginal orifice (VO) . 1/ In cooperation with the Louisiana State University and the University of Wisconsin. 2/ The insemination apparatus, exclusive of microscope and anesthe- tic equipment, can be purchased from the Department of Economic Entomology, King Hall, University of Wisconsin, Madison, Wis. -u- FigTire 2 illustrates the internal portions of the reproductive tract -ftlth the side toward the observer removed. The dorsal and ventral plates and the sting are not shown, but portions of the walls of the sting cham- ber are seen at the right. A fold across the anterior floor of the sting chamber loosely separates a region called the bursa copula trix from the sting chamber proper. The vagina (V), throxogh its vaginal orifice (VO), and the bursal pouches (BP) open into this region. There are two bursal pouches, their openings lying at the side of and below the vaginal orifice, Only the left pouch is shown in figure 2. The position of their openings in relation to the vagina, when the queen is properly mounted for insem- ination, is shown in figure 1, BP. They are easily found with a dull probe. Sometines, when the queen is poorly mounted, a beginner might mistake a bursal pouch for the vagina. The spermathecal duct (SPP) from the spermatheca (not shoflm) enters the vagina anteriorly from above. Just below the opening of this duct is the valvefold (VF.)* ^ large tonguelike 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 bet7/een the ''agina ?nd the median oviduct (SK and K) with a valvelike action. The paired oviducts (POV) enter the median oviduct anteriorly. Each paired oviduct leads to an ovary (not shown). They are large fluted structures capable of great expansion for the temporary storage of sperm after mating and of eggs in a laying queen. In figure 2 the reproductive tract is extended. During insemination the queen is held in such a way that the vagina is collapsed, and the valvefold often appears to lie just inside the vaginal opening (fig. 1, VF). Laidlaw found the diam.eter of the vaginal orifice to vary from 0.65 to 0.68 mm., and the average diameter of the oviduct orifice to be 0,33 nmi. These diameters are important considerations in syringe construction. A detailed knowledge of the anatomy of the drone reproductive organs is not necessary for the mastery of the insemination technique. A des- cription of their structure and function is given by Laidlaw (2). Dur- ing natural m.ating the penis everts, and the reproductive fluids are ejaculated probably more or less simultaneously vdth the eversion. The semen, a cream-colored fluid containing the sperm, passes out first and is followed by the mucus, Tvhich is more viscous, pure white, and coagu- lates after ejacul?tion. Further details about the ejaculation process are given under Insemination Procedure. Queens just returned from the mating fli/?ht have been studied by a number of investigators. According to Laidlaw (2), who reviewed the earlier vfork, most of the semen is found in the oviducts, some is in the vagina, and some has already migrated into the spermathecal duct and spermatheca. Parts of the penis are found in the sting chamber buried in mucus, which also extends into the folds of its Walls. Since the penis apparently does not enter the vagina, the queen probably lowers the valvefold during the mating act to permit the semen to pass. -5- In order to gat the s«Bi«n in its natural position by artificial insemination, the valvefold lauat be pushed ventrally to permit the point of the syringe to pass into the median ovlduot* If the syringe enters only the mouth of the vagina, the semen presses the ralvefold against the median oviduct and is forced back etround the syringe and out into the sting chamber. The vagina is not easily distendable, but the ovi- duots expand to take oare of a large quaatity of ssaen* BQVvIPMEHT AHD CONSTRUCTION OP INSTRDMEHTS Microscope and Light The major equipment needed to perform the artifioial-lnsemination operation is Illustrated in figure 3, and the aenipulating apparatus with instruments in place is shoim in more detail from the side of the operator in figure 4« The binocular dissecting microscope should preferably be one provided vith a revolving nosepieoe or other mechanism making adjustment from low to high power easy* The low power should give a ma^ifloation of about 6 diameters, and the high power about 20 diameters* A single inter- mediate magnification can be used* 1'^en two powers are used, sperm is taken into the syringe imder low power azid injected under high power. ▲ still higher magnification will be fovmd useful in making and measuring instruments* An attached lamp that always illuminates the focal point of the microscope is a great convenience. Carbon Dioxide Equipment Carbon dioxide serves as an anesthetic* The equipment for its appli> cation is illustrated in figure 3. This gas is obtained in cylinders from wholesale grocers or similar supply houses. To reduce the high pressure in the cylinder, a regulator is provided which permits adjustment to a delivery pressure of 4 to 5 pounds per square inch. A needle valve permute adjustment of the flow of gas to a very fine stream. A rubber tube carries the gas to the queen holder by way of a three«-way stopcock, which permits diversion of the stream of gas while the queen is being mounted, without disturbing the needle'-^alve adjustment. Another line leads into a Jar, in which queens are given additional anes the ti cations to be described later. The Manipulating Apparatus The manipulating apparatus is a stand (fig* 4) on which the queen holder (QH), the syringe {Sj , and holding hooks (VH and STH) are mounted in such a way that they oeja be adjusted or manipulated* It is essentially the same as the apparatus developed by Nolan (5^), with modifications which make more accurate adjustment possible* The stage (ST) is made of a piece of l/4-inch iron plate, 3 inches wide and 11 inches long, raised at each end by a piece of wood thick enough to permit the microscope base to slip under the stage* The weight of the stage gives stability to the apparatus. Two upright 3/8»inch brass rods threaded at the bottom end are screwed into the stage at the points illustrated, and to these rods all other parts are attached. The rod at the left of the operator is 3 inches high and the one to the right 3 1/2 inches. These rods are 4 I/2 inches apart center to center. -6- The movmt for the queen holder Is Bftde of wood, and consiats of a queen-holder movmting block (QHMB) att&ohed to a horitontal strip (HS). The atrip is l/2 by 5/4 by 6 inches, with a hole bored near eaoh eixd to that it can slip up euad down on the two posts and with set screws to hold it fast at any desired level. Near the right-*and post is a hori- zontal slot about 2 inohea long* M long bolt passing through this slot and also through the block makes it possible to adjust the block at any desired angle* The queen holder (QB) fits into a hole in this block, and a set screw holds it seourejy in place. A wide groove is out on the att- sorew side of the hole, and a piece of leather tacked to the top of the block fits into this groove preventing the set screw from scratching or breaking the queen holder* It alec keeps the queen holder in place by light friction until the set screw can be tij^tened* The set screw works in a hole bored Just snail enough for the threads to take hold* Since the mountings for the syringe (S), the ventral hook (VH), and the sting hook (STH) are essentially similar, a detailed description of the syringe mounting will suffice for all three* A block of wood, the syringe mounting block (SMB), is bored so that it will fit snugly over the post as illustrated* On the left side of the post this block is divided by a vertical saw out, and a S/l6»inch bolt is provided with which the separated parts can be pulled together if necessary to increase friction on the post* Thia bolt may have a wing nut, or one aide of the divided block may be bored to a analler diameter* In any caee the head of the bolt must be flat enough not to interfere with the movement of the syringe* On the other side of the post the syringe holder (SH) is attached to the block* The syringe holder is simply a piece of sheet metal cut and bent into the shape of a box l/2 by 1/2 by 1 l/2 inches* A l/8 by 1-inch bolt 1/2 inch from one ezul holds i;he bottom of this box to the block* This bolt projects about 1/2 inch back of the block, and a piece of coil spring is put on under the nut so that the ffiotion between the box and the block can be increased or decreased by tightening or loosening the nut* In each end of the box a hole is bored of sxioh a size that the syringe will slip through easily but not loosely* A oxirved piece of clock mainspring (SP) slipped in between the syringe and the side of the box holds the syringe in place* The ciirvat\u*e of this spring can be ad*« Justed to permit the syringe to slip in and out easily but still stay in place when released* The ventral hook and stizig hook are mounted in the same way, except that the boxes for them are s/8 by s/S by 1 l/2 inches* It is important to keep the syringe and the hook handles clean and polished with oil or a hard wax to make them slip easily* If the wooden parts are treated with wax, they will not expand and contract excessively with changes in humidity* Queen Holder The queen holder (fig* 6) is a modifioation of the type devised by Jas* I* Hambleton and first used extensively by Nolan (£) • It is a tube slightly constricted at one end, into which a long stopper fits snugly* The queen is made to back into the holder until her abdomen protrudes -7- from the small mxtd eind then is held in plaoe by the stopper. Both parts are made of Lucite, a transparent plastlo* Ihe stopper is also a tube, which permits a stream of oarbon dioxide to flow gently over the queen, keeping her anesthetieed during the insemination operation* The drilling and polishing of Luoite are disoussed later under Plastio Syringe* Dimensions are given of the holder used suooess fully by the authota* There oan be some flexibility of dimensions, but it is well to remember that the thoraxes of most wellMdeveloped queens range in diameter from 0«196 to 0*205 inoh* The opening at the end of the holder may be from 0*18 to 0*19 inoh in diameter* The 5/l6»inoh taper is adequate to permit only the last three segments of the queen to protrude from the holder* This taper may be obtained by stretching the plastio tube after heating, as desoribed under oonstruotion of the syringe tip, or by drilling with a suooession of drills* Several small grooves should be out with a three- oomered file on the inside of the holder at the tapered end to faoilit&te the esoape of gas* The stopper diameter muet be very near the diameter of the holder (0*267 ineh) to move in and out of the holder with sufficient friction to remain firm at axiy plaoe« If the stopper loses friotion through wear, its diameter may be iaoreased with a light eoat of plastio oement or laoquer* The slif^t protuberanoe at the front end of the stopper has four addition- al lateral outlets (not shown in diagram)* This protuberanoe aids in adjusting the queen in the holder and protects her antennae from being ■tashed* The enlarged knob at the posterior end holds the rubber tubing onto the stopper* The stream of oarbon dioxide frooi the oylinder passes throu^ the stopper, is dispersed by the five openings at the end of the stopper, and flows over the queen and out the end of the holder* If faoilities are not available for making the plastio queen holder, a glass one is easily made by drawing 9««ni* glass tuW.ng to about 4 mm* entside diameter, finding the right inside diameter for the small end, bretking at this point, and grinding off the edges. Since oommeroial gXMi tubing varies in sise^ it is aeoessary to seleot a pieoe of the proper inside diameter fron the 9««n* sise* The stopper is a pieoe of spongy paper, suoh as paper toweling, ro-lled ever a matoh stiek, whioh is later pushed out to make a thiok* walled tube* If the tube is of the right sise, friotion with the walls of the glass holder keeps it in plaoe* It is made long enou^ to projeot from the glass holder when the queen is in plaoe, and additional paper is glued onto this end to make it taper* If preferred, the oarbon dioxide supply tmbe oan easily be slipped on and off the tapered end at eaoh operation, and the threewway stopoook elimiaated* Syringe The syringe is a modif ioation of the instruments used by other workers in this field* It ooasists of a small tube inside of whioh a tightly fitting plunger draws in and expels the semen* Two types of syringes are being used* Glass Byringe> For th« glass lyringf (fig* 6) & gl&st tub« i« employed as a plimg^r barrsl* This tube will hereafter be referred to as the glass tip or simply the tip* A brasi spring wire §9xy9 as a plunger, and • meohanioal peneil, as reooooaeaded by Solan {i), proridet the mechanism for Boring the plunger* The glass tip is attTohed to the end of the pencil housing^ and the plunger takes the plaoe of the lead* A number of special tools and materials are needed for a oonstruotion of this type* For drairing oapillary glass tubing an ordinary bunsen burner will do, but a blast burner is a great ooxxv^enienoe where both gfts and air are avtil* able* A gasoline blowtoroh has been found rery satisfaotory* A Jewelers* fine«grain glass^grinding wheel is needed for grinding off glass tips to the right diameter* Suoh a wheel oan easily be mounted on a shaft and made to turn slowly by means of a belt azid motor* ^e lower part of the wheel should dip into a tray of water to keep it flooded as it tvu*ns. The cooling action of the water prevents ohipping of the glass* A special gpis burner is needed for produoing a fine flame for use in forming the tapered end of the glass tips* This bvimer oan be a piece of e-^nuxit glass tubing drawn to an inside diameter of 0*5 mm* at one end* The burner is attached to flui ordinary gas outlet, which is adjusted to preduoe a flame about 5 mm* in diameter* A compound microscope equipped with a micrometer eyepiece and baring a magnification of about 60 diameters is useful in measuring the diameter of the glass tips as well as other small parts of the insemination equip* ment while under construction* Some of these measurements oan be made with an ordinary adorometer* If a compound microscope is not arailable^ a miorometer eyepiece oan be used in the dissecting microscope with a magnification of about 60 diameters* A number of small tools that oan be obtained fr(»i Jewelers* supply houses are needed* They include No* 6 cut files of various shapes, small flat-nose and pointed-nose pliers, and rods and triangular slips of fine- grain Arkansas stone* The end of a rod is ground on a coarser stone to a oone-shaped tip, -nhioh is used in grinding the inside edges of the oapillary tubing* These tools are also used in making the holding hooks and vaginal probe* A supply of No* 26 B, and S. gage wire should be on hand for making plungers. Several kinds of wire will do, but spring brass is preferred because it is stiff, yet solders well, and does not corrode easily* Some mechanical penoils are better adapted for syringe making than others. One having a round housing and using a 4-inoh load has been found most satisfaotory* This type (fig. 6) has a stationary, hollow metal core with a slot running its entire length* The lead and lead holder slide in this core, and a spiral made of steel wire or a steel strip encloses almost its entire length* Projections on the lead holder extend through the slot into the spiral, and as the spiral ic turned the holder slides, moving the lead in or out of the pencil* A number of ohflinges must be made to adapt a pencil to its new use. The friction that keeps the lead at the adjusted level must be eliminat- ed* Usually the pencil must be taken apart to accomplish this. Since the friction is caused in different ways in different makes of pencils, the exact procedure cannot be described. The point of the pencil is sawed or filed off sufficiently to let the lead holder project. If the housing is hexagonal, it should be replaced with a round tube, preferably of solderable material. The lead holder should be filed to a smaller dia- meter to allow more play between it and the core, or it can be replaced entirely with a piece of wire bent and filed into shape, «hen the pencil is reassembled the spiral should turn without friction. For the plunger a piece of 26 B, and S, gage brass spring wire is selected which has no kinks to prevent a tight fit. Any gentle bend resulting from the wire being wound on a spool may be straightened by pulling the wire between thumb and forefinger a number of times, but sharp kinks are difficult to remove and should be avoided. One end of the wire is ground off squarely, and the burred edges are polished. Then after the wire has been cut to the proper length, the other end is soldered into the hollow end of the lead holder. The length of the plung- er will depend upon the construction of the pencil and on the length of the completed glass tip. It should be slightly longer than the tip, etnd long enough not to slip out of its upper end i^en withdrawn as far into the pencil as possible. If there is still some bend in the plunger, it should be attached so that the back of the arch will rub against one of the sides of the core. Such a position will prevent the arch from push- ing the end of the glass tip against the spiral and possibly breatking it, and at the same time will prevent the end of the plunger from slip- ping out of the core groove when drawn into the pencil while no glass tip is present. To make a good glass tip requires some practice and the exercise of proper care. The first step is to draw out a supply of capillary tubing. Ordinary 6-mm. soft-glass tubing has been found very satisfac- tory for this purpose. Long sections of fairly uniform bore can best be obtained by heating a section of tubing about 1 inch long, drawing slowly at first until the center of the section is of about the right diameter, and then drawing more rapidly to a length of 3 to 4 feet, "^ith 26 gage (0.41-mm,) wire the inside diameter is tested until a piece of nearly uniform bore about 60 mm. long is found which fits closely. The glass is broken at the point where the end of the wire fitted most tightly, and this end is made the inside end; the larger end becomes the point of the syringe. The inner edge of the small end is ground under water by twirling on the pointed Arkansas stone, so that there will be no sharp edge to scratch the plunger and gradually decrease its diameter. The plunger should not be inserted from this end until the glass tip is completed and ready for mounting, and then only under the microscope, because the glass is easily broken if the plunger is not inserted perfectly straight. -10- The large end of the tip is drawn to a smaller diameter to form the point of the syringe. To do this heat a spot near the end in the small gas flame eind draw the ends to a diameter of somewhat less than 0*27 mms Sometimes two or three attempts must be made before a satisfactory product is obtained. The drawn section is then scj-atched with the sharp edge of a stone, broken off at the proper point, and groxmd back to an outside diameter of 0,27 mm* The outside edge is ground off lightly on the grind- ing wheel and the inside edge by spinning between thxmb and forefinger on the pointed stone under water, or with a drop of water covering the point. The point is then polished until it appears perfectly smooth when ma^iified about 60 times, by twirling the tip on the end of a soft piece of wood rubbed with jewelers* rouge, or by holding it against such a piece of wood moimted on the end of a moror shaft* The completed tip snould De 35 to 50 nm« longa To attaoh it to the penoil, put it orer the plunger and draw it into the penoil until it sticks out about 25 an., then glue it into plaoe with an aoetone-soluble cement sush as is used to build model alr- plsnaes* This oement is easily dissolred when it beoones neoessax^ to replace the tip* The proper dlsoMters at the point of the glass tip are of utmost Importanoe* The outside diameter must be small enou^ to permit passage into the oriduot, and yet the inside diameter must be large enough to per- mit semen to be taken up easily axid quickly. The outside diameter should not be oyer 0*5 mm«, and 0*27 mm* is usually satisfactory. The inside dia- meter should not be less than 0.15 mm* These limits differ slightly from the dimensions recommended for the plastic syringe* When made as desoribed with 26-gage wire (0«41 mm.), the glass tip has a oapaoity of 1 mm*' for every 7*6 mm* of length* If a 27-gage (0*56 mm*) plunger is used, the oapaoity is 1 mm*^ for every 9*6 mm* of length* Gk>od instruments have been made with both sizes. Tips of smaller diameter are too delicate and have too small a capacity to be practical* Larger ones are more difficult to finish at the point with a large enough inside diameter and a small enough outside diameter* Plastio syringe. The plastic syringe is made of Lucite and brass* Most of itw parts must be made on a lathe* The Lucite tip is superior to glass because it is not easily broken, sharp edges are eliminated, and it can be machined and drawn to the desired diameter and bore. Threaded re- placeable tips can be readily changed and are easily cleaned. Lucite does not have the surface hardness of glass, and consequently will wear slowly. It softens when heated* (h*ease and oil can be removed with hexane, naphtha, methanol, or mild soap and water. The machining qualities of Lucite and brass are similar. Since this plastic is a poor conductor of heat, it is necessary to prevent overheating when drilling, cutting, or polishing. Drills should be lubricated with mineral oil or an oil solution C2 percent of soluble oil in water)* Fre- quent removal of chips is necessary when deep holes are drilled, and the holes are filled with the oil solution at each removal of the drill. Sharp drills and proper lubrication give uniform and polished inside walls to the tip, which result in good suction without a lubricant. -11- All the syringe parte are shown In flgxire 7, and the struotural details of the tips and plvmger are given in figure 8* Sinoe the neaaore* nents and details of construotion of the housing^ turning screw, oouplings, and supplemental rod are of secondary importanoe, they will zu>t be described. Their approximate else can be ascertained from the pict\u*e# The uniynrsal Joint between the turning screw and the supplemental rod allows the metal plunger to move within the tip without rotating. Lack of rotation reduces wear ejod gives better suction* The tip is made from a Lucite rod of 0«1875-inch diameter* This rod is placed in the machine and drilled to a depth of about 1 inch with a No. 72 (0.025 inch) drill, A smaller drill--No.79 (0,0145 inch)— provided with a long shank is then used to drill an additional 0*1 inch in the bottom of the 0.025-'inch hole* The outside diameter is then turned down to 0*164 inch, and the end threaded for a distance of about 0.26 inch with an 8-<32 threading die* Before the rod is removed from the machine, its diameter between the threaded portion and the end of the drilled portion can be reduced to the approximate site of the main portion of the tip (0*085 inch). The end of the tip containing the small hole is then rotated on an electric soldering iron imtil it becomes soft enough to stretch. This 0*l»inch section with the 0«0145-inch bore is then stretched to a length of 0.2 to 0*25 inch and, while still held taut, is hardened in cold water* If the rod is properly heated, the 0*025-inoh hole is not distorted* The 0*0145-inoh hole becomes a gi*adual taper from that diameter down to less than 0.006 inch* The end of the tip is then filed off square, and the outside diameter reduced to 0*012 inch* No* 2 cut files can be vised to reduce the diameter to near sire, but the polishing should be done with 9/0 production finishing paper* The final polish may be obtained by rub- bing with moistened cigar ash* The plunger can be made from brass spring wire of 0.025* inch dia- meter* It must be straight and miiformly round without snarp edges. The butt end is soldered into a 0*0625-inch brass rod which has been threaded to screw into the supplemental rod of the syringe* Holding Hooks The function of the holding hooks is to hold the sting chamber open and the sting back out of the way dxu*ing the operation* The ventral hook (figs. 1 and 4, VH; fig, 9) fits over the T«ntral plate* It is constructed of a piece of No* 24 gage brass wire slightly flattened at the end, and bent as illustrated in figure 9* The distance from the bottom of the U to the tip is 2.5 mm*, and the distance between the inside walls of the U is 0*37 mm* The tip of the ventral plate fits into the U-shaped portion so that the plate remains in a vertical position as it is pulled ventrally by the hook* The dorsal or sting hook (figs* 1 and 4, STFj fig. 9) is designed to pull the entire sting mechanism dorsally. 1 similar instrument was used by Laidlaw (2^). It has an enlargement at the end, which fits into the -12- trlangular area between the basal portions of the sting lancets, and extends vmderneath them (fig. l) • Figure 9 gives' a side view of this Instrument at A and a top view at B^ from the angle Indicated by the arrow In A. The enlarged tip Is 0»77 mm« wide. The thinnest part of the stea is 0*08 mm« when viewed from above and 0*17 mm. when viewed from the side* The stem is bent to fit the queen parts. This Instru&ent is made from wire about 0«92 mm. in diameter, and filed down to the proper shape with jewelers' files. The enlarged tip represents the end of the wire filed off at an angle. All rough edges of both the ventral and sting hook are polished first with a fine stone and then with Jewelers* rouge. Both hooks are soldered to the ends of brass rods 3/l6 inch in diameter and 5 inohes long, irtiloh serve as handles and fit into the sheet-metal boxes* Other Equipment Other items of equipment needed for the operation are a pair of for- ceps, a sting depressor, a dish of water for cleaning the syringe, a vagi- nal probe, and a container for treating drones with chloroform* The sting depressor is simply a dull-pointed dissecting needle. The probe (fig. 9) is an instrument used to push the valvefold down- ward. It is a piece of No* 24 gage brass wire bent at rigjit angles 4 mm* from one end* This end is flattened to a thickness of 0*1S mm* on a plane with the main stem of the instrument and polished down to a width of about 0*19 mm. The probe is bent slightly near the tip, so that the main part of the wire does not obstruct the view when the tip is Inserted into the vagina* Drones are made to ejaculate in a l-by-4-inch glass vial containing paper soaked in chloroform wadded in the bottom* A stopper fastened to the table so that the vial can be put onto it in a horizontal position is a great time saver* INSEMINATION PROCEDURE Adjusting the Flow of Carbon Dioxide The end of the carbon dioxide supply tube is iamersed in water and the flow of gas adjusted to a slow bubbling. It should not be faster than .is necessary to keep the queen completely anesthetlred. Experience will soon show just how much this should be. The tube is then passed through the queenholder mounting block from below, the stopper attached, and the three-way stopcock in the supply line turned, to prevent gas from passing through the stopper while the queen is being mounted. Preparing the Syringe To function properly the tip of the glass syringe must be filled with water, which acts as a lubricant and increases the suction of the plunger. Vlater is firdt drawn into the tip by withdrawing the plunger* -13- Then, while a flngor is held against the end, the plunger is pushed out again until the air around it has been entirely replaced by water* A 5«4im* column of water is left at the end of the plunger* The plastic syringe can be prepared in much the same way as the glass syringe when a lubricant seems necessary* Ordinarily the water will be present after the rinsing operation described later. The plunger should be pushed out as far as it will go* Tap water has been found satisfactory, but in some localities distilled water may be better* Physiological salt solution must not be used with the glass syringe, because it cannot be thoroughly rinsed and, when the water dries up, the salt residue freezes the plunger in the tip and corrodes the syringe parts* Preparing the Queen The queen is made to walk into a tube similar in site and construc- tion to the queen holder* When she reaches the partly closed end she begins backing up, and if the queen holder is quickly put in place she usuedly backs into it* The stopper is pushed in after her until her abdomen protriides from the sme^ll end of the holder and she cannot move about readily* For best results only about the last three segments of the abdomen should protrude* The three-'way stopcock is turned so that carbon dioxide will flow throu^ the queen holder* The queen breathes heavily for a few minutes and then gradually becomes quiet* If her abdomen expands abnormally, the gas is being forced into her abdominal air sacs, an indication that the flow is too strong* Unless very severe this action seems to do no harm, although it makes insemination difficult* The holder is withdrawn into its mounting block by pulling on the carbon dioxide supply tube, and tightened in place by the set screw* The dorsal part of the queen should be at the right of the operator* The queen holder should be about 30° from vertical, with the upper end leaning to the right (fig* 4)* The queen is usually quiet by the time she is completely mounted, and the holding hooks can be put in place. This is done under low-power magnification* First one hook and then the other is inserted into the sting chamber, and the abdominal plates sure 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 the sting hook is left in this position to prevent umiacessary drying of the delicate tissues while the syringe is being loaded. Filling the Syringe The microscope is withdrawn slightly and the syringe placed in its box, with care not to break the tip* This is done with greatest safety by depressing the spring with forceps while the syringe is being Inserted* The microscope and syringe are then so adjusted that the end of the syringe is in focus* -u- A drone is anesthetised by droppingMm in the vial already described. His abdomen contracts and usually the penis partially everts, as illus- trated in figure 10, A, He is then made to evert more completely by being squeezed between thumb and forefinger. There is great variation in the degree of ever s ion, the distribution of semen and mucus, and the amount of semen ejaculated* Bversion usually stops at the stage illus- trated in figure 10, B^, which is about two-thirds complete. As the pro- cess of eversion proceeds, the cream-colored semen passes out first fol- lowed by the thicker white mucus* If the eversion process can be stopped at just the right point, a drop of pure semen will be found at the tip with all the mucus left inside. IVhen this ideal situation exists it is very easy to take up the semen. Usually, however, at least some of the mucus comes out after the semen, and the two are distributed on the penis in various arrangements. Often the semen is spread so thinly over the mucus that it is difficult to take up. It is best to see that an ample supply of drones is available and to use only those that evert and ejaculate most satisfactorily. This is especially important when individual matings are being made, so that the maximiam amount of semen is obtained from the single drone used. Movement of sperm causes the semen to spread in a thin layer over the mucus and to mix with it until too thick to be taken into the syringe easily. This mixing also takes place while the drone is partially everted, ^t is, therefore, important that drones be utilized as soon as possible after anesthetization. Without abdominal contraction semen is rarely obtainable, but when the abdomen contracts without partial eversion the eversion can often be completed by pressure, and a good amount of semen obtained. The ejaculated drone is brought near the tip of the syringe with the left hand and the plunger withdrawn slightly to make an air bubble. The surface of the semen is then made to touch the point of the syringe at about a 45-degree angle. If the syringe is raised slightly after contact has been made, the semen will adhere to it and flow toward it as the plung- er is withdrawn. This procedure helps to avoid the mucus, which is more viscous and will not flow so easily as semen.. Mucus is too thick to pass through the end of the syringe easily and will stop the passage of semen. "kVhen this happens, the plunger is pushed out until the passage is clear- ed and then the. taking of semen is continued. By moving the syringe about, the mucus 6an be skimmed of practically all its semen covering. Semen is taken from as many drones as necessary to fill the syringe to the desired point. As the syringe is filled, it will be noticed that the suction pulls some of the water from around the plunger, thus increasing the amount above the air bubble. Later, as the plunger is pushed out during injec- tion, this water passes back around the plunger. This action will be negligible with a well-made tip. When the plvinger is poorly fitted, however, it is sometimes withdrawn as far as it will go before the desired amount of semen has been drawn up. In this case the plunger can be pushed down again while a finger is being held over the end. Such a tip is a time consumer and should be replaced as soon as possible. Some water should remain between the end of the plunger and the air bubble to pass back around the plunger as the semen is injected; otherwise the air bubble, or oven some of the semen, may pass around the plunger. -15- The function of the air bubble is to prevent the mixing of semen and water and to mark the boxmdary between them so that diluted semen will not be injected. As injection proceeds semen adheres to the side walls and mixes with the water which follows, so that in the absence of an air bubble it is difficult to distinguish between pure and diluted semen, iihen a series of inseminations is made without the syringe being cleaned, as is possible with the plastic syringe, the air bubble is superfluous o Injection As soon as the syritige is filled, the point is moistened to prevent clogging by drying of semen and to lubricate it. The syringe is moved into position over the queen and the microscope pushed forward so that the queen is in the field and the magnification changed to high power. The sting hook is then drawn dorsally xintil the sting chamber appears as in figure 1. This act stretches the loose membranes so that the vaginal orifice, and often the valvefold, are visible o It also stretches the dorsal wall of the vagina so that the syringe can slide along it into the median oviduct. Vifith the left hand the probe is inserted into the dorsal part of the vagina and the valvefold pushed ventrally, until the point of the syringe has passed beyond. Then, as the syringe is pushed in farther, the probe is removed • The syringe should be inserted no farther beyond the valve- fold than is necessary for satisfactory insemination (about 1 to 1.5 mm.). The plastic tip can be inserted deeper than the glass tip on account of its more gradvially tapering point. If it appears that the syringe carries tissue in with it as if caugjht on the end of the syringe, then the point has not hit th6 median oviduct and should be withdrawn and reinserted, possibly after some readjustment of the holding hooks. Tfiith the syringe in place the plunger is slowly pushed downward. If the semen moves down the barrel easily and does not leak out around the point, then the syringe is in proper position and the injection can proceed rapidly. If the glass syringe is used, great care must be exercised that the plunger never reaches the cone-shaped point. The plunger of the plas- tic syringe, on the other hand, can be pushed to the end of the larger bore without danger of breakage • The syringe is now withdrawn from the queen, and then from its box, the spring being depressed with forceps so that the syringe slips out easily without danger of the point breaking. Removal of the queen from her holder completes the operation. Cleaning the Syringe The glass syringe should be cleaned immediately, by alternately draw- ing in and expelling fresh water until the liquid inside the syringe is relatively clear. ^Vhen the plunger does not fit closely, a little semen left in the liquid may help to increase suction. „,„•»■'»*«' -16- Regardless of the care exercised, a coating gradually accumulates on the inside walls of the syringe, and finally interferes with the passage of semen. This coating can be removed with a piece of wire 0»002 inch in diameter slightly bent at the end. Flakes that are too large to pass through easily can usually be removed by drawing back the plunger slowly so that water passes around and behind them and then push- ing out very suddenly so that they will be carried withthe stream of water. Several repetitions of this procedure will usually clear the barrel. The glass tip can be cleaned by dipping in a hot solution of an alkali such as sodiiim hydroxide or common lye, which dissolves the depos- its; however, the alkali must be thoroughly rinsed out before the syringe can be used again, "hen using alkali it is best to withdraw the plunger 15 to 20 mm. and leave the tip full of air, then to draw in a small quan- tity of the hot alkali. Later the alkali can be forced out and the rins- ing done without any contact with the plunger. The plastic syringe need be cleaned only when it is to be stored or when the plunger becomes too tight. Cleaning is simple. The tip etnd plunger are unscrewed, and water is drawn in and out with the plunger with such force that usually all obstructions are easily rinsed out. Sterilizing the Syringe Fnen a quick change is made from one genetic type of sperm to another, it is necessary to sterilize thd syringe. The glass syringe is sterilized by rinsing it thoroughly and then dipping it in boiling water deeply enough to submerge part of the pencil as well as the exposed part of the glass tip, so that the inside end of the tip also becomes heated. If several syringes are available, a fresh one oeua be employed for each kind of drone used in a single day. The plastic tip is sterilized by quickly drawing in and expelling 95 per cent ethyl alcohol, and then thoroughly rinsing with water immediately, because Lucite is slowly soluble in alcohol. ESTIMATING THE NUMBER OF SPERMS One criterion of successful insemination is the number of sperms that reach the spermatheca. A fairly good estimate can be made by the following method : Items of equipment needed include a counting chamber such as is used to count blood corpuscles, a 50-cc. burette, a medicine-dropper pipette, a small glass dish, a pair of sharp-pointed forceps, and a dissecting needle. The pipette should have an inside diameter of about 1 mm. at the tip. For the glass dish an individual saltcellar almost as deep as wide, having a capacity a little over 10 cc, and having a rounded bottom somewhat smaller than the diameter of the rim, has been found to be just the right size and shape. To dissect out the spermatheca one must tear off the last segment of the abdomen by grasping the last ventral plate with forceps. The spermatheca is usually found imbedded in the tissue inside this removed segment. It is a sphere about 1 mm. in diameter and appears rough and white because of the network of tracheae which covers it completely, ^''hen the tracheae are -17- removed, the spermatheca is fovuad to be smooth and transparent in a virgin queen and milky-white to cloudy-cream in mated queens, depending on the degree of insemination. The spermatheca is placed in the dish suad 1 cc# of tap water added from the burette* Then the spermatheca is broken with the sharp-pointed forceps and needle, the sperm teased out, and the empty skin removed. The sperm is dispersed by alternately dravring the water into, and expelling it from, the pipette about twenty times, or until all lumps have disappeared, Then 9 cc« of water is added to make a total of 10 cc. and the sperm again thoroughly dispersed with the pipette. A drop of this mixture is quickly placed in the co(jntiag chambero ^'hen the sperm number seems to be small, a dilution to only 5 cc« or less c&n be used. The sperms are counted under a compound microscope with a magnification of 60 diameters, and against a dark field, which makes the sperms stand out as white filaments. From the number counted in a certain volume the nvimber in 10 cc. is calcu- lated. The sperms usually appear as almost headless, slightly curved, fila- ments about 0.27 mm. long, but they may be coiled or looped into various shapes, su%h as small circles, spheres, or like the numerals 6 and 8. It is important that the dispersion be continued no longer than is absolutely necessary and that the drop be placed in the counting chamber quickly before any great amount of settling or adhesion of sperm can take place. It is also important that the pipette be kept clean to prevent lumps of sperm from sticking to its walls. In some localities a physiolog- ical salt solution may be more satisfactory than tap water. Distilled water is not recommended. There is, of course, considerable chance of error in making counts from such a small sample. Nevertheless, this method is certainly more accurate than judging by appearance of the spermatheca and the viscosity of the contents, as has been done by previous investigators. Greater accuracy can be obtained by counting a larger sample. All sperm counts given in this manual were made by diluting individual matings to 5 oc. and other matings to 10 oo* and counting the sperms in 0«8 mm*^ The number of sperms in the seminal vesicles of drones can be estimated by the same method. A window is carefully out into the dorsal wall of the abdomen eind the seminal vesicles are very gently cut off where they join the mucus glands. If this is not done very carefully, the muscles of the seminal vesicles will contract and some of the sperm will be lost. After being placed in water, the muscles are made to contract by pricking and mashing, and what sperm is not forced out in this vmy is released by tearing the seminal vesicle to pieces. THE USE OF CARBON DIOXIDE Carbon dioxide not only serves as an anesthetic, but also stimulates queens to begin laying (Mackensen 4^) . Proper treatment with this gas causes virgin as well as inseminated queens to begin laying practically as soon as naturally mated queens, whereas without such treatment only about 20 percent start earlier than 30 days after emergence. -18- At least two anesthetizations of 10 minutas duration spaced a day apart are necessary to start egg laying. These may or may not be accom- panied by insemination* ^n anesthetic treatment without insemination is given by placing the caged queen in a convenient container through irtiich carbon dioxide is flowing (fig. s). If the queen is to begin laying at the normal age (8 to 11 days after emergence), the second treatment must be completed before the seventh day, and preferably before the sixth day, because she begins laying 2 to 6 days after the second treatment. Although treatments given as early as the second and third day are effective, they do not cause laying to start earlier than the normal &ge« PROCEDORES FOR TAKTOUS TIPES Of KATINGS AND RESULTS TO BE EXPECTED Inseminations should be made from the fourth to the tenth day after emergence. They have often been made earlier than this (second and third day), but at times with poor results. At the other extreme, queens can be inseminated at any age if they have not begun laying, but if not made to start soon after the tenth day, they will be balled and mistr^ted by the worker bees, and the percentage and quality of the laying queens will be reduced. As much as 10 mm.^ of semen has been given in one operation with suc- cess, but because the percentage of sperm that reaches the spermatheca de- clines as the load increases, it is better to give several small insemina- tions than a single large one. Except for individual matings, a load of 2 .5 to 4.0 mm. 3 has been found very satisfactory. Results are also less variable when several inseminations are made than when an equal amount of semen is given in a single insemination. It is best to allow 2 days between inseminations for clearing of semen from the reproductive tract, although often inseminations on successive days have been quite successful. The operation should be performed as rapidly as possible to avoid unnecessary exposure of the sperm. Individual Matings Often it is desirable to mate each queen with a single drone. In this case it is important that the maximum possible nimiber of sperms be obtained from the single drone used. Therefore, well-matured, well-nourished drones should be available, and only those used that seem to ejaculate perfectly and to yield a large amovmt of semen heavy with spenn. The best drones yield nearly 1 mm.' of semen. Individual matings are usually made only in genetic experiments in which it is imperative that the spenn come from but a single drone. Therefore, if a series of such matings is to be made, the syringe must be sterilized between operations to prevent contamination with sperm previously used. In individual matings the percentage of laying queens is usually high, and most of them will produce some worker offspring. However, so few sperms reach the spermatheca that the supply does not last long, and such matings are only made when absolutely necessary. One addi- tional carbon dioxide treatment must be given to start egg laying. -19 Qn« Insemination With Spenn From Sevoral Drones When neither individual mating nor long productive life is desired, several drones from the same source can be used, making one injection con- sisting of at least 2»5 inm,^ of semen. Viith this procedure it is almost certain that none of the queens will begin laying as partial or complete drohe layers, and if kept in nuclei most of them will last a season with- out becoming drone layers* One additional carbon dioxide treatment is necessary to start egg laying. Two or More Inseminations To fill the spennatheca sufficiently to carry a queen through a season in a large colony, two or more inseminations must be made, each consisting t>f 2»5 mm.' of semen* An additional carbon dioxide treatment is not neces- sary* Hundreds of queens mated two or three times have performed as well as naturally mated queens* A still greater number of sperms cern be made to reach the spermatheoa by giving three or four inseminations with 2*5 mm*' of semen* They are given on alternate days beginning on the fourth day or, in the case of foxir inseminations, they may be started the third day to reduce the likelihood of interference with the beginning of oviposition. Considerable variation can be expected in the percentage of laying queens obtained* In one group of 95 queens inseminated three times, 83 per cent started laying; however, results as low as 60 percent can be 4xpected occasionally* In general, the more inseminations made the more chances of injury and consequent loss. Practically all queens that sxirvive to laying age will begin laying* Table 1 compares the number of sperms in various artificially inseminated and naturally mated queens* These data are taken from Several experiments* Note that the coefficient of variation decreases as the number of insemina- tions is increased, and that it is lower in some of the inseminated queens than in those naturally mated. Note also that the minimiim in the group inseminated three times is higher than the minimum in the naturally mated queens, and that the average and maximum in the group inseminated four times are nearly as great as the corresponding figures for the naturally mated queens* The performance of artificially inseminated queens compares very well with that of naturally mated queens* In 1945 queens inseminated artificially three times were compared with queens of identical parentage mated naturally at an isolated mating station* Surplus honey produced was about the same for the two types of matings, as already reported by Roberts (j) , Brood production and brood quality were not significantly different, but survival to the end of the season was somewhat lower in the artificially inseminated group* -20- Table 1«- A ttompasison batwaan tha numbar of sparm in tha sparmathaoa of naturally mated queans and artificially inseminatad quaans inseminated as indicated* Treatment Number of queans Number of sperms in spermatheoa (millions) Average Range Coefficient of variation Artificial insemination: Semen from one drone 17 0,87 0»22 - 2,24 51 Semen from many drones (2.5 mm.5) Once Twice Three times Four times 11 2*97 9 4.11 10 4.85 11 5.52 1*28 - 4*41 2.05 - 5»65 3»71 - 5,80 4.66 - 6.79 35 29 16 10 Natural mating 35 5,73 3.34 - 7.35 18 THE ABSOLUTE CONTROL OF PARENTAGE The purpose of artificial insemination is tha control of parantaga, and the virgin queens and drones used are reared from eggs laid by selected breeder queens which are already mated. The virgins are reared from fertil- ized eggs and inherit from both the breeder queen and the drona with which she mated, whereas the drones develop from unfertilized eggs and inherit only from the breeder queen. These are the desired breeding individuals, but occasionally other types of individuals appear in the hive, with which they might be confused. These types are (l) drones emd queans reared from unfertilized aggs of laying workers and (2) quaans reared from the unfertilized eggs of the breeder queen. Only tha drones are a serious menace to pure mating, but all types will be discussed because they must be considered in the interpretation of genetic experiments. The number of of f«typa drones and queens depends largely upon the number of laying workers in the hive which varies considerably with race, season, and hive conditions. Queenlessness for any length of time will cause some woricers to start laying, and they may continue to lay after the colony has been made queen right. Laying workers are apparently mora common in early spring, when the hive is populated mainly with old, ovar^ wintered bees. %ey are very oommon among the Cape bees of South Africa (Jack 1) , but in the bees of the United States and tha European races they are relatively rare. Under conditions where they could be positively identified, 13 off- type drones were found to emerge among about 5000 drones of the right type reared in a full-sized colony, v^ioh is at the rate of 0*26 par cant* Drones have been reared under controlled conditions for a number of saasonPyand although the percentage of off-type drones has usually not been determined by actual count, it is estimated not to have exceeded 0.26 par cent. Often -21- none were found among several thousand drones. They can be recognized and avoided if the breeder queen is introduced to a colony of bees of a distinguishable color. For example, if a queen of a yellow strain is introduced to bees of a black strain, her sons and daughters will be yellow and the sons of worker bees will be blaok. If a selected breeder queen is supported entirely by her own worker daughters, it is not so important to guard against the off-type individuals, because all descend from the breeder queen and her mate, and therefore repre- sent only the desired germ plasm. Often, however, it is unpractical to wait for a queen to produce her own supporting population. The other types of undesirable individuals, queens reared from \mfer- tilized eggs of either queen or worker, are called impatemate because they have no male parent. These queens are quite common in some races. Among Cape bees of South Africa laying workers develop readily and produce female offspring in abundeince when the hive becomes queenless (Jack _1 ) • In other races they have been considered to be rare or not to occur at all, but Mackensen (_3) has proved their occurrence in three American strains of bees representing the Caucasian and Italian races. He found female progeny in the brood of 21 out of 50 virgin queens tested. Of the larvae grafted from one queen producing a high percentage of female offspring, 0*85 per cent developed into queens. Impaternate females also occur in the related insect Habrobracon juglandis Ashm., and Speicher (_8) has presented evidence that they develop from diploid eggs which arise from patches of tetraploid tissue in the ovaries. The frequency with which impaternate daughter queens of laying workers are likely to be encountered in rearing queens by the usual methods has not been determined. If the percentage of impatemate females developing from unfertilized eggs of queens. (0.85 per cent) can also be taken as the percentage of laying worker eggs that develop into females and the rate of occurrence of off-type drones (0«26 per cent) is taken as the percentage of laying worker eggs in the hive, the expected rate of occurrence of queens of this type csui be calculated to be 0.000022 per cent, or 1 in 4.5 million. Obviously they are so infrequent that precautions need not be taken to avoid them. If it should be desirable, the method described for off-type drones could be used, Impaternate queens developing from eggs of the breeder queen inherit only from her. In ordinary breeding work there is no way of recognizing and avoiding them* They probably do not occur among the larvae grafted at all because fertilization would lead to triploidy and probably death, and the larvae ordinarily used for grafting develop from fertilized eggs. CARE OF QUEENS To provide the most natural conditions queens are not kept in nursery cages prior to insemination, as recommended by some workers on artificial insemination, but introduced as cells and maintained in nuclei until they begin laying* Excluders are placed, over the hive entrances to prevent the loss of queens by mating flights. As soon as possible after emergence, -22- rhile the queens are still recognizable as newly emerged, one wing is clipped to prevent flight and the thorax is mariced with a spot of color. A lacquer called Libralao mixed with the desired pigment is suitable as a marking fluid. A solution of celluloid in acetone is also a satisfac- tory vehicle for the pigaent* The queen is taken directly from the nucleus and returned immediately after insemination, while she is still completely motionless from the effect of carbon dioxide. TShen she is in this condition, the bees are less likely to ball her than if she is active. In warm weather she is dropped into the nucleus, but in cool weather she is left in the cage in which she is transported. The cage is opened and slipped between the top bars of the frames* REARING AND CARE OF DRONES It is not always easy to obtain large numbers of drones of the desired parentage. During the spring they can usually be obtained in abimdanoe from any queen, because they are naturally produced at this sea- son, but later in the year it is sometimes almost impossible to obtain any drones at all from some queens. Sometimes, even though the queen will lay in drone comb, the workers refuse to rear larvae to loaturity, "hen a heavy flow is on, the drone comb is filled with fresh nectar so that the queen does not have a chance to lay drone eggs. Drones are most likely to be obtained out of season if conditions similar to those existing during the swarming season are created. Pollen and sugar sirup can be fed if necessary, and a crowded condition created either by adding bees and brood or by reducing the size of the hive, ^'■hen the nectar flow is heavy, empty combs can be provided to help keep the drone comb clear. If drone combs are extracted gently, the thin nec- tar can be removed without destroying eggs and young larvae that might be present in some of the cells. A sure way to obtain drones is to rear them from drone-laying queens, which can be produced by treating virgin queens Ydth carbon dioxide, as already described. These virgins would, of course, be daughters of the selected breeler queen. The following routine has been used successfully for a number of years to rear and maintain large numbers of drones of controlled parentage in yellow strains I A five-frame nucleus is used as a drone-rearing colony. It is made up with three combs of brood from black selected Caucasian stock, a comb of honey, and a drone comb, which is placed in the middle of the nucleus. Extra pollen is provided by sprinkling dry pollen pellets into the combs and spraying with water. The yellow breeder queen is introduced with a push-in cage and released 2 or 3 days later, depending on how well she is accepted. The nucleus is stocked with 3 l/2 pounds of black bees added from a screen cage which covers the top of the nucleus hive. They are forced to go through an excluder, which sifts out drones as they leave the cage. The bees are kept confined for 2 days. Pollen is added occa- sionally, and the nucleus is sometimes fed very lightly to help stimulate the queen. -23- As soon as the first drone oomb is well filled, emother drone oonb is substituted for one of the brood combs. This is usually about a week or 10 days after the queen has ooamenoed laying. After another week or 10 days the first drone comb is removed to a drone nursery colony and re- placed with a third drone comb. In this way sometimee three or four drone combs can be filled within a month after laying starts. Special precautions are taken to prevent strange yellow bees, which might develop into laying workers, from drifting into the drone-rearing colony. The colony is established at least 100 yards away from the queen- yard and frequent examinations are made to find and kill any yellow bees that might have drifted in. If other drone— rearing colonies are kept near by, they are removed before yellow bees begin to fly from them. The drone nursery colony is placed in or very near the queen yard, so that drones from it are conveniently available for insemination. It is kept separated from other bees as much as possible to prevent drifting in of yellow bees* A l-story 10-frame standard hive is provided with 5 or 6 combs of brood and 4 pounds of bees of black stock, added from a screen cage through an excluder, together with some honey and a great excess of pollen. The bees are confined for 2 days, A screen top is placed under the cover, so that ventilation can be provided by lifting the cover slight- ly when the excluder at the entrance becomes crowded with drones trying to get out. The bees rear their own queen, which remains a virgin because she cannot get out of the hive to mate • Her presence prevents the develop- ment of laying workers. More brood is added later to provide a continual supply of young bees. If the nursery colony is so situated that yellow bees drift in, the drone combs are removed after 24 days, and all other drone brood is killed at 10-day intervals. These precautions are taken to prevent sons of lay- ing workers from maturing, and are unnecessary when the entire worker population is of a body color distinguishable from the drones being nursed by the colony. All manipulations of the nursery colony are made early in the morning while drones are not normally flying. OfteA it is necessary to work the colony in a portable screen cage to prevent stray drones from drifting in. After long confinement or when the presence of excluders has kept them out of the hives during the night, drones will often fly in the morning. The screening of bees for rearing and nursery colonies serves two purposes. It eliminates superfluous black drones which would crowd entrances unnecessarily and also prevents the accidental addition of a queen. For these reasons the procedure has become a matter of routine not only in stocking these colonies but also in stocking nuclei. At the age of 8 days drones become sexually mature and try to leave the hive* At this age the maximum number of sperms has accxanulated in the seminal vesicles, but the drones often do not ejaculate well until they have been trying to get out for a nximber of days. The feces that accumulate -2A- during confinement seem to be an aid rather than a hindrance. Its pre- sence seems to increase the pressure inside the abdomen when the muscles contract, assuring good eversion of the penis and ejaculation, it has been noticed that drones whose flight has not been restricted by confine- ment to the hive will ejaculate better and in greater numbers after they have been kept in a cage for a day or two vdth workers, sugar sirup, and pollen. Drones do not live so long when coni'ined as when they are able to fly naturally. In the nursery colonies some of .them die soon after they reach the age of 25 days, and few reach 35 days. Three important requirements of the drone nursery colony are ample bees, ample pollen, and queenlessness. Drones need the care of worker bees, ^hen kept in cages and provided only with pollen, water, and sugar syrup, they soon die, but when about twice their number of worker bees are added, they live to sexual maturity and contain a good quantity of usable semen, rthen pollen is omitted drones live for some time, but very few sperms develop. >iew contributions to the technique of instrumental insemina- tion of queen bees. Jour, icon, int, 22: 944-954, illus. -27- Figure 1, — VjLew of sting chaaber of queen properly opened for insemtnatiouj BP. Opening of bursal pouches ; HP, dorsal plate; fifi* queen holder; ST, sting; STH. sting hook; VF, valrefold; Vg, ventral hook; VQ,, vaginal orifice; VP, ventral plate. -28- SH K VF VP Figure 2. — Reproductive tract of queen daudad of the -ovanies, extended, with Bide removed to show relation of valvefold to median oviduct and vagina: BP, Bursal pouch; K, keel of median oviduct; POV, paired ovi- duct; SH, shelf of median oviduct; SM. sting membrane; SPD, spermathe- cal duct; V, vagina; VF. valvefold; VO, vaginal orifice; ^, vaginal passage. (From La;L,dlaw) . Figure 3. — Complete insemination equipment, showing manipulating apparatus under microscope with queen in place ready for injection of semen, jar for giving additional carbon dioxide treatments, and carbon dioxide cyl- inder with pressure regulator attached. -29- Figure U, — UanlpiLLatlng apparatus viewed fxx)m operator's side, showing mounting of queen holder, syringe, and holding hooks: HS, Horizontal strip; ^, queen j QH. queen holder; QHMB. queen-holder mounting block; S, syringe; SH, syringe holder; SMB. syringe mounting block; SP, spring; ST, stage; STH, sting hook; VH> ventral hook. 1^4. iNi 01* 1875' — » 257" l«--5f6-»l ~-~OUT.DIA, .362* QUEEN HOLDER 2>6. 01* X3ei' STOPPER Figure 5. — Structural details of the queen holder, -30- o ^% O -P CO a a o o a II EH _ to ml -31- Figure 7. — Parts of plastic syringe. (From top to botton): Turning screw with supplemental rod, two Lucite tips, two plungers, and housing. LUCITE TIP .025" DIA :^ 0145" DIA; --INS DIA .008" 'outs DIA 012." 25" END OF TIP PLUNGER Figure 8. — Structural details of the Lucite tip and its plunger, -32- VENTRAL HOOK STING HOOK 0J7mm/ 0.08mm/ Figure 9, — Structural details of vaginal probe, ventral hook side view, and sting hook with A, side view, aind B, top view from the direction indicated by the arrow X. The arrow also marks the point where the end begins to enlarge. ■33- SEMEN — MUCUS- Figiire 10, — Stages of the aversion of drone's penis: A, partial eversion usually encountered after anesthetization with chloroform; B, a more complete eversion usually obtained by squeezing the abdomen, with semen and mucus exi:)osed.