Dr. H. Allen Sylvester

Dr. T. E. Rinderer

USDA, ARS, Honey-Bee Breeding,

Genetics & Physiology

Research Laboratory

1157 Ben Hur Rd.

Baton Rouge, Louisiana 70820

INTRODUCTION

The main characteristic of Africanized bees which must be considered in any discussion, but particularly a discussion of identification, is that they are hybrids. The extent and affect of this hybridization may vary in different locations and situations but it is present. Electrophoretic analysis of enzymes (Sylvester 1986) and morphometric analysis (Buco, et al. 1986) of African, European and Africanized populations of bees have clearly shown that the Africanized bees were hybrids. The result of this hybridization is that mixed or intermediate colonies exist. Also, since bees live in social colonies with multiply-mated queens, sister workers will make up subfamilies, each descended from a different drone, which may be Africanized, European, or intermediate. The workers in any one colony may also be descended from more than one queen because of queen replacement or drifting.

Nevertheless, an accurate and reliable method of identifying Africanized bees in the field is needed. Through research being done at the USDA, ARS, Honey-Bee Breeding, Genetics & Physiology Research Laboratory in Baton Rouge, Louisiana, this laboratory developed a method for quick identification of Africanized bees, based on research done at the University of California at Berkeley by Dr. Howell V. Daly. This identification system was named FABIS – Fast Africanized Bee Identification System.

The analyses used in FABIS are statistical procedures which compare body-part measurements of unknown samples to chosen groups of known samples (base populations) and give probability estimates that the unknown sample has body-part measurements similar to those of the base populations. For FABIS, the samples in the 2 base populations (Africanized and European) were identified based on their known history and field behavior and this identification was confirmed by the method of Daly et al. (1982).

Forewing length is the one most important measurement in identifying an Africanized bee. This step takes about 20 minutes per colony.

Where forewing length alone can not correctly identify a sample, a group of two measurements and one weight has proved to be the best. They are: the lengths of the forewing and femur (Figure 1) and the “fresh weight” of the bee. The fresh weight is the weight of the bee with its abdomen (gaster) removed, and it is the second most important measurement in FABIS. These measurements take about 1 hour per colony.

Where a correct fresh weight cannot be measured, the samples can be processed as for fresh weight and then dried to yield dry weight. Dry weight is less discriminating than fresh weight, but is still useful. However, a reasonable extra effort to obtain an accurate fresh weight will be well repaid by the increased discriminating ability of fresh weight.

FABIS is thus a process for quick, simple, field screening of large numbers of bee colonies. FABIS has been tested with data from other Africanized and European colonies, and the accuracy of the method has been verified for European bees from the U. S. and Africanized bees from Venezuela.

The sensitivity of this technique necessitates a warning. Ideally, users should verify that the European bees in their area are similar to the European bees in this study before these procedures are used to detect Africanization. If the European bees are not similar, especially if they are smaller, locally collected baseline data may be used to develop new discriminant functions or at least be considered in evaluating results. Such new functions would be more appropriate to the users’ needs.

This version of FABIS is discussed in more detail in a scientific article (Rinderer, et al. 1986b). Fresh weight is called “wet weight” in that article. An earlier version of FABIS was discussed by Rinderer et al. (1986a) and by Sylvester and Rinderer (1986).

PROBABILITIES AND MEASUREMENT CHOICES

Choosing which probability level to use and interpreting the results obtained will depend on the purpose for which the samples are being analyzed. The simplest ease is for samples from a population which is known to fit the FABIS baseline (i.e. where the range of forewing lengths in the sampled populaticn is within the range of lengths of the FABIS base population) and where the total cost due to destroying the sampled colony would be small. In such a case, forewing length or fresh weight alone at a probability of Africanization of 0.99 or greater (PA 0.99) would be sufficient to identify a sample as Africanized. Where the population baseline is unknown, a lower PA cutoff point may be better for early samples with any colonies suspicious of Africanization analyzed by additional FABIS measures or other methods for confirmation. Where there would be significant costs as a result of identifying a sample as Africanized, such analyses should include two or three FABIS measures and be confirmed by comb measurements, field behavior or Daly’s full morphometric analysis, before a colony is declared Africanized.

Because of the improved accuracy of these techniques, there are several choices of identification procedures. Depending upon needs, capabilities, and equipment availability, any of three single measurements can be selected for preliminary identification. Forewing length is the best and fresh weight is second best. Because freezing and transport reduce the value of fresh weight, where fresh weights are used, only freshly killed bees can be expected to give satisfactory results. When frozen and transported, all samples become heavier, apparently because of the freezing, thawing, and associated water condensation. These conditions can lead to misidentifications. Dry weight overcomes these technical difficulties in maintaining accurate weight. Thus, in spite of its lower discriminatory power, dry weight may be better than fresh weight in large programs or where transportation of samples is difficult.

For all single characters, at least a few samples may remain unidentified. Any of the combinations can be used to identify such samples. The best pair combines forewing length and fresh weight. Its major disadvantage is that it requires equipment to measure both lengths and weight, but it is the simplest and fastest. Forewing and femur lengths may be desirable where only lengths can be measured. This is the second best approach. Where dry weight was used as an initial screening tool, the best addition would be forewing length.

Femur lengths add to the power of forewing length, fresh weight, and dry weight. We therefore present forewing length plus femur length plus dry weight and forewing length plus femur length plus fresh weight, because large programs may have a need for these analyses. The most precise of these (forewing length plus femur length plus fresh weight) gives the best separation of Africanized and European populations using these simple methods.

SAMPLING PROCEDURES

Sampling procedures will vary somewhat depending on the purposes of the collector. One constant requirement, however, is that each sample should come from one swarm, colony or hive and not be a composite sample. The FABIS probabilities (Table 1) are based on an average of measurements of groups of bees from a single source rather than on measurements of individual bees. Distinctly different individual bees in a sample will not be detected by this method. Bees of unknown origin (e.g. bees at a feeding station) may be analyzed by these procedures but the results must be examined at the individual bee level. If all bees from such samples are Africanized, one or more colonies of Africanized bees are in the area. If all the bees from such samples are European, no Africanized bees were detected in the area. If some of the bees appear Africanized, then one or more Africanized colonies may be in the area. In this last case, positive confirmation can only come from direct sampling of the kept and feral colonies.

For baseline or survey sampling, particular attention should be paid to swarms, feral colonies, and unmanaged colonies. They are the most likely to be of unique local types or be newly arrived Africanized colonies. Generally, it will be desirable to have as broad a survey as possible. That is, samples should be taken from as many different areas as possible, then from as many beekeepers or locations in each area as possible. Except for total colony or stock certification-type sampling, it is not desirable to collect more than a few samples from any one beekeeper, regardless of the number of colonies owned.

The number of bees collected should be more than are needed for the procedure(s) planned since some bees are often unsuitable due to damaged wings, etc. The same bees can often be used for more than one procedure; that is 3 groups of 10 bees can be weighed and then one of these groups can have the wings and femurs removed for measurement. However, for large sampling operations, it is more efficient to collect enough bees that different bees can be used for each procedure.

EQUIPMENT NEEDED

The equipment needed includes: 1) fine-pointed forceps; 2) 22×40 millimeter (mm), #1, microscope slide coverslips; 3) clear plastic tape; 4) 35 mm, plastic, slide mounts; 5) a 35 mm slide projector; 6) an ocular micrometer (e.g., Thomas Scientific #6588-M-80)(2); 7) a clear plastic 0.5-meter-long scale; 8) a metric balance that is accurate to 0.01 gram and 9) a hand-held calculator. A dissecting microscope is very helpful when learning the proper dissection procedures. If available, it will also be helpful later to examine dissected parts to see if they are ready to mount. The slide mounts should be a brand that is open and then snaps shut for mounting, such as Polaroid® (2), because the coverslips are very thin glass that is easily broken. They cannot be bent for mounting. For measuring the projected images, a solid surface is needed so the images stay in focus during measuring and a light-colored, reasonably smooth surface is needed for ease in focusing. The simplest way to meet these requirements is often to fasten white, roll paper onto a wall (Figure 4). Depending on its lens, the projector may need to be up to 20 feet from the wall. The projector should be on as secure a support as possible to minimize measurement errors due to movement of the projector. The lens on most projectors is curved to match the curvature of 35 mm slides and so produce an image entirely in focus. The coverslips are flat which produces an image which is not entirely in focus. Then focus adjustments are necessary as measurements are taken across the image. A flat lens is available which should produce a focused image from edge to edge and reduce time spent in adjustments.

To make the measurements, a custom-made 0.5-meter-long scale is very helpful. This is simply a clear plastic stick, 0.5 meter in length, with 1 mm graduations marked, and with the 1 cm units replaced by numbers 1/5 as large (e.g. 20 becomes 4.0). This scale is then read directly to give actual body part lengths in hundredths of millimeters. In practice, the only points which need to be numbered are 0.0, 8.0 to 10.0 for forewings, and 2.2 to 2.8 for femurs. The simplest way to produce such a scale is to purchase a standard 0.5 meter, clear plastic ruler (e.g., a #M-111 made by C-THRU(2)) and renumber the above points (Figure 2).

PROCEDURE

Calibration

An ocular micrometer is fastened to a 22×40 mm coverslip with tape so that the micrometer scale is in the same focal plane as will be the bee body parts. This coverslip is placed in one of the 35 mm slide mounts and placed in the slide projector. A calibration image is projected on the wall and the projector moved until the 10 mm scale on the slide-mounted ocular micrometer produces a 0.5-meter image on the wall. This is a 50 to-1 magnification, so that measurements can be taken quickly and easily divided by 50. Or, if a custom-made scale is used (Figure 2), the measurements are read directly from this scale by measuring the length of the projected image.

Calibration should be checked frequently to ensure the system remains accurate. We recommend that recalibration be done hourly. Before any other action is taken as a result of a sample scoring as Africanized, the calibration should be checked to confirm that the measurements are correct.

Forewings

Prepare the 22×40 mm coverslips by fastening 2 together on the short side with a small piece of clear tape acting as a hinge. Carefully remove the forewing from the bee, so that the entire forewing is obtained, if care is not taken, the wing often breaks, leaving the base on the bee, and the correct length cannot then be measured. On fresh samples, the forewings can usually be simply pulled from the bees, which have been killed in an insect killing jar or by being briefly frozen. Lay 5 forewings between 2 coverslips. Two coverslip mounts will be needed to mount the 10 forewing per sample. A skilled person may be able to place 10 wings between a single pair of coverslips. Then close the pair of coverslips so that the wings are separated and their ends are visible, and fasten the other ends together with a small piece of tape. Forewings are not fastened directly to single coverslips with transparent tape because the adhesive on the tape blurs the wingtip and it often cannot be seen clearly.

The 22×40 mm coverslips fit into standard, 35 mm slide mounts (Figure 3). Project these slides onto the wall, measure the images to obtain 10 forewing lengths (Figure 4) and calculate the average forewing length for the sample.

Femur

One of the hindlegs from each of 10 bees is removed, prepared, as shown in Figure 1 and mounted between coverslips in the same way as forewings. The trochanter must be removed from the femur so that the length to the tip of the femur, which is covered by the trochanter, may be measured (Figure 1). Also, the leg must be bent as shown in Figure 1 so that both ends of the femur can be clearly seen. The end of the femur which is covered by the trochanter is fragile and often obscured by remaining pieces of the trochanter. Therefore, if possible, it is often easier to remove the trochanter while using a dissecting microscope so that it can be seen when all parts of the trochanter are removed.

Fresh Weight

The fresh weight is determined by removing the abdomens (gasters) only, by carefully pulling them off with fingers or fine-pointed forceps. Also, any pollen pellets are removed from the legs, the weights of 3 groups of 10 bees are measured on a balance to the nearest 0.01 g, and the average weight per 10 bees is calculated. In order to obtain accurate weights, the bees should be immediately frozen with dry ice in portable coolers and transported to the lab. The weights should be measured as soon as possible.

Dry Weight

In some cases, the bees cannot be collected, transported and processed carefully enough to yield an accurate fresh weight. If so, they can be prepared as for fresh weight, dried for 24 hours at 60ºC (140ºF), and then weighed to yield a dry weight.

Analysis of Measurements

After the average lengths and weights have been calculated, they will be compared to values in Table 1. For single measurements, the calculated average value is used directly to determine the probability the sample is Africanized (PA). If the calculated value is less than or equal to the table value for 0.99 (e. g., for forewing, 8.968 = 0.99), the sample has a PA of 0.99 or more (PA~ 0.99). If the value is larger than 8.968 but equal to or less than 8.991, the PA is less than 0.99 but greater than or equal to 0.95 (0.99> PA>-0.95); and similarly for 0.90. If the calculated value is greater than the table value for 0.90, the sample is clearly European in any case. In most cases, the cutoff point to use is PA>0.99.

Where 2 or 3 measurements are made, the average values interact and cannot be used directly. Simple formulas are given where the calculated averages are substituted in the proper formula, a result (function) is calculated, and the result is compared to the table values given in the same way as for single measurements.

ACKNOWLEDGMENTS

Our thanks to Lorraine Davis and Dan Winfrey for their technical assistance in this research.

FOOTNOTES

(1) In cooperation with Louisiana Agriculture Experiment Station.

(2) Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.