THE P-3 and EST LOCI in the HONEYBEE APZS Mellzferal

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 enzyme are controlled by two genes, P-3 and Est, with two alleles each. The frequency 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 incompletely dominant alleles; the heterozygote P-3S/P-3F shows only an intermediate 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 individual 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 inseminated 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

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. the same test applic4 to the two subspecies indicates that they have differcnt P-3 allele frequencies (x2 = 168.3; df = 2; P < 0.001). The frequency of the P-3' in Apis mdlifera adun- sonii (African bees) is 0.995 while for the populittion of Apis mdlifrwz ligu.s/ica ! Italian bees) it is 0.531. Thr?Est .sys/rm:A zymogram of the supernatant from worker and drone pupac. produced by the open-mated queen 67-5-68 indicated the prescwce of two csterase variants. Figure 2 shows the forms dcitected in drones and workers. Mobilities of the esterases wcre designated Est-S and Est-F. The results are listed in Table 1 in the last six lines of (lata; they are consistent with the hypothesis of two codominant alleles (IWand E.s/~)at the same locus. The workers present three phenotypes produced by combinations E.s/"/l:.s/" (Figure 2-l3). Est"/E.s/" t

A B C D E FIGURI:2.--Starch gel electrophoresis pattrms of thr three tliffrrerit esterase activities. The pattrrns corrcspond to the Renotyprs: (A) P EsP'/Estr; (13) P Esls/Essl"; (C) d Est'; (D) P I.d"/E.st"; (E)J ESP.I'hr arrow shows the direction of migration. ISOZYMES IN BEES 73 7 (Figure 2-D), EstF/EstF(Figure 2-A) and the drones show two forms produced by genotypes EstS (Figure 2-E) and EstF (Figure 2-C) . The same 75 colonies used for determination of the frequency of the P-3 alleles provided the material for the analysis of the esterase alleles. The results are pre- sented in Table 3. No difference was found between the populations or subspecies (P > 0.20). The frequency of Est" in these combined populations is estimated to be 0.972. Only the Italian population can be called polymorphic for the P-3 locus if the limit of 0.010 established by MORTON,KRIEGER and MI (1966) is accepted. But the frequency of EstF in both subspecies is 0.028, and this locus should there- fore be considered polymorphic under any definition. Some of our colonies were headed by double heterozygous queens, P-3S/P-3F; EstS/EstF,namely 84-4-68,174-2-68 and 25-10-69, which were inseminated by one drone each, of the following respective genotypes: P-3";EstF,P-3";Est" and P-3';EstS. This allowed us to determine whether these two genes were linked. The progeny of these queens segregated as is shown in the Table 4. The results indicate no linkage between the two loci.

TABLE 3 Phenotypic and genotypic frequency in jour populations of tum Apis mellifera subspecies Est system

Phenotypic frequency Genic f.mquency Number of Number of Locality colonies individuals Ests/Ests Est~@st~Estp@sta Est# ESP Araraquara(I) (African bee) 26 1M 0.971 0.029 0.OOO 0.985 0.015 Araraquara (11) (African bee) 42 168 0.934 0.066 0.000 0.967 0.033 Sub-Total 68 272 0.976 0.024 Rio Claro (Italian bee) 3 12 1.000 0.000 0.000 1.000 0.ooo Ribeirgo Preto (Italian bee) 4 16 0.820 0.180 O.Oo0 0.829 0.171 Sub-Total 7 28 0.914 0.086 Total 75 300 0.943 0.057 0.000 0.972 0.028

TABLE 4

Resulis of gonic segregation of three queens P-3S/P-3F ;EstS/EstF in order to test linkage or independence of the tum loci

Frequency of the gonic segregants Number of the queen Genotype of the ovules 84-468 174-2-68 25-10-69 Total P-3F ;Est" 6 7 5 18 P-3" ;Est" 9 9 5 23 P-3" ; Est" 5 4 10 19 P-3" ;Ests 8 8 8 24 738 M. A. MESTRINER AND E. P. B. CONTEL

Prof. WARWICKESTEVAM KERR directed the Doctoral Dissertation. Prof. C. D. MICHENER criticized the manuscript. Dr. L. S. GONEALVESand Mr. J. F. CAMARGOcarried out the artificial insemination and bee management. Prof. E. W. CASPARIread this paper and made valuable sug- gestions.

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