THE P-3 and EST LOCI in the HONEYBEE APZS Mellzferal
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THE P-3 AND EST LOCI IN THE HONEYBEE APZS MELLZFERAl MOACYR ANTONIO MESTRINER AND EUCLEIA PRIM0 BETIOLI CONTEL Department of Genetics, Faculdade de Medicina, University of &io Paulo, 141OO-Ribeiri?o Preto, SP.,Brad ABSTRACT Data for Apis mellifera indicate that the P-3 proteins and one esterase en- zyme are controlled by two genes, P-3 and Est, with two alleles each. The fre- quency of the P-3 alleles is different in the two subspecies (udunsonii and Zigus- &a), that for P-3F in Italian bees being 46.9% and in African 0.5%. The frequency of Ests is 2.8% in both populations. The Est locus has two codominant alleles and the locus P-3 has two incom- pletely dominant alleles; the heterozygote P-3S/P-3F shows only an inter- mediate band. The two loci are not genetically linked. EES provide good material for genetic work; while studies have been made on their proteins (LENSKY1967; GILLIAMand JACKSON1972) and isozymes ( TRIPATHIand DIXON1968, 1969) little work has been reported on the genetics of such macromolecules. Four qualities make bees suitable for such studies; a) artificial insemination and numerous beekeeping techniques have already been developed, making these insects readily controllable; b) it is possible to obtain great quantities of material since populations of a colony may vary from fifty to 150,000 workers; c) bees are large enough to furnish material not only for indi- vidual electrophoresis but also for electrophoresis of individual tissues and organs; d) bees are a totally haplo-diploid genetic system. This last property is the field of work of many researchers in this laboratory so that any discovery by one is im- mediately used by others. A preliminary report on the data collected before March, 1969, has been pub- lished (MESTRINER1969). MATERIALS AND METHODS Our material can be divided into two lots. One, consisting of 25 colonies with artificially in- seminated queens, was used for a study of the Mendelian aspects of the problem; all 25 queens were Italian (Apis mellifera Zigustica). The second lot of 75 colonies, consisted of 68 containing African queens (Apis mellifera adansonii) and 7 Italian queens; from each colony, 4 worker bees (a total of 300) were used for determining the gene frequencies in Italian and African bee populations. The colonies of each subspecies were derived from two different localities, so that each subspecies is represented by two populations. Using the formula Ne = 15 C/7 (KERR 1967) where Ne = genetic active number and C 1number of colonies, our sample was taken from a population with Ne= 160.7. This work received support from SBo Paulo State Research Foundation (FAPESP), Brazilian Research Council (CNPq) and Rockefeller Foundation. This paper represents part of the Doctoral Dissertation of the senior author. Genetics 72: 733-738 December 1972. 734 M. A. MESTRINER AND E. P. R. CONTEL Artificial insemination was carried out according to the techniques developed by LAIDIAW (1949) and MACKENSEN(1947), with the small modifications made in this laboratory hy J. CAMARGOand GON<ALVES(1968, 1971). Recently we began using the technique of C. A. CAMARGO (1972) using coconut water (pH 7) in the syringe. Our queens were numbered according to the system of IAIDLAW(19.54). Thr method of Doo- little for rearing qurens was used (as descrikl in LAIDLAWand E~KFRT1950). Drones were pro- duced hy Riving colonies a frame with drawn drone foundation. Ten-clay old drones and five- to seven-day old queens wrre used in the artificial crosses. Electrophorrsis was carried out on (lark eyed, white to cream colorrcl pupac.. Thus there was no need for isolating the hers in screenrd rages to avoid contamination by hers from other hives and the intestines were empty and contained thus no pollen proteins. Workrr or drone pupae were each put in a test tube 7.5 x 80 mm, containing 0.1 (for work- en) ancl 0.2 (for drones) ml of distilled water; each pupa was homogenized with a glnss rod at room temprrature (25 to 32OC). Then it was rrntrifugrtl for 10 min at 3,000 rpni. Ahout 20 pl of supernatant from each tube was used for elrctrophorrsis (SMITIIIES3955) with the huffrr sys- tem of POUI-IK(1937). Starch grls were put on 6 x 9.5 x 200 mm plates, and rrceircd 14 samples at a time. exposed for 3 hr, ilt 10°C and 2 mA per cm (a total of 40 mA). Before staining, the gels wrrr srrtionrd into two parts: one was stninrcl with Amido Black 10 B. the othrr was incu- hatrtl for 30 min using the trchniques of VVRIGIIT(1963) for detecting esterase activity. RESULTS AND ANALYSIS The P-3 system: Three main zones, intensely stained with Amido Black 10 R. were found in the supernatant of both workers and drones after electrophoresis. According to their migratory rates. these zones were designated P-1.P-2 and P-3. Preliminary analysis of workers and drones produced by queen 147-1-68 (naturally mated) indicated the existence of individual variation in migratory rate in the P-3 protein. Figure 1 shows all forms detected: Figures 1-A. 1-R,I-D + \ A B C D E FIGURE1.-Photographs of the threr main zones of protein migration in starch grl elrctro- phoresis of honey her pupae. The patterns for the third zone (from ln4ow) correspond to the genotypes: (A) 9 P-3s/P-3s; (R) 9 P-3F/P-3'; (C) 8 P-3F; (D) p P-3s/P-3p; (E) 6 P-3s. The arrow shows the direction of migration. ISOZYMES IN BEES 735 TABLE 1 Crosses made to demonstrate the inheritance of the protein P-3 and esterase enzyme variants in pupae of bees offspring Type of cross Workers Drones Number of colonies Queen Drone PJ@/PJU P-3@/P-3f P-3F/P-3f P-3s P-3P P-3S/P-3S P-3s 84 0 0 56 0 P-3S/P-3S P-3" 0 56 0 28 0 P-3S/P-3F P-3s 29 27 0 43 41 P-3S/P-3" P-3F 0 93 103 42 42 P-3F/P-3" P-3s 0 56 a 0 56 P-3"/P-3" P-3" 0 0 a4 0 a4 Est#/EstbJ Estu/EsP Esif/EstF ESP ESP 3 EstS/EstS Ests 84 0 0 56 0 3 EstS/EstS Estp 0 84 0 56 0 4 EstS/EstF Est8 39 45 0 41 4d 8 EsP/EstF Est" 0 107 117 62 50 2 EstF/EstF Est8 0 56 0 0 56 2 EstF/EstF EstF 0 0 56 0 56 are workers and I-C and I-E are drones. The pattern shown in Figure I-D is the heterozygous female. In Table 1 the results of twenty crosses and 924 individual electrophoreses of workers and drones are listed in the first six lines of data. Drones and workers of each line are the progeny of queens with the genotypes listed in column 2. All the data are consistent with the hypothesis of two incompletely dominant alleles. As shown there, the P-3 proteins can be found in the following genotypes and patterns: workers P-3s/P-3S (Figures I-A) ; P-3"/P-4" (Figure 1-B); P-3'/P-3" (Figure 1-D) and drones P-3" (Figure 1-C) and P-P (Figure I-E). The intermediate electrophoretic mobility for the heterozygous P-SS/P-3' seems to indicate a specific type of interaction of subunits of these two forms (P-gSand P-3F).A mixture of equal parts of P-3 fast and P-3 slow proteins does not produce the heterozygous pattern but produces two independent zones. This uncommon feature suggests that a hybrid dimer is produced in the workers and the association of the subunits is not random. No heterozygous forms have been found in drones, a finding which agrees with their haploid constitution and with determination by one single gene. ALLEN,MISCH and MORRISON(1963) have found that in Tetrahymena pyri- formis heterozygous for isozymes of the P-I locus, some stable subclones show only the P-I-A band, while others have the P-I-B band and still others only a band with intermediate migratory rate. They suggested that the intermediate band represents clones in which both alleles are active and form only the hybrid protein. In order to obtain an estimate of allele frequencies, 75 colonies with open- mated queens were used (14 workers per hive, 300 bees, 600 genes). The results 7 36 M. A. MI-STRINIIR ANI) 1:. P. n. CONTEL TAR1.E 2 Phmotypic rind genotypic frequrncies in four pOpU~/i~ifJ?ISof two Apis mellifrra subspecies 1'-3 system Phenotypic frcqiiency Genic frequency Siitnlwr of Sunilx-r of lmality colonies individunlc P-3s/P-38 P-3"/P-3F I1-3v/F'-3r P-3" P-3' . ~~ ~ ____.___~ __~__~__ Araraqtrarii f I) 1 Afrirarl twr) 26 10F 1 .OW 0.000 O.000 1.000 0.000 Araraquilrii ( I I ) t African twr j 42 168 0.082 0.01 8 0.000 0.991 0.0~ Total ti8 272 0.9% 0.OM Rio Clnro (Italian twr) 3 12 0.083 0.584 0.333 0.37'5 0.62.5 Rihrirh Prrto iItalian hrr) 4 16 0:50O 0.37.; 0.125 0.687 0.313 Total 7 28 0.531 0:Uic) :ire prescnted in Table 2; data for Iigustim and cidcinsonii are shown independent- ly. A contingency x" test indicates that the two populations of each subspecies have identical frequencies of the P-3 alleles; however.