MOLECULAR EXCLUSION CHROMATOGRAPHY OF CRUDE VENOM AS AN AUXILIARY TOOL TO IDENTIFY HYBRID HONEYBEE POPULATIONSAbstract
A comparison among the profiles of molecular exclusion chromatography in Sephadex G 100 column of venoms from Apis mellifera adansonii and Africanized honeybees revealed unique peaks which might be used to identify these populations. The venoms from hybrid populations resulting from the reciprocal mating of Apis mellifera adansonii and Africanized honeybees presented unique peaks, probably resulting from a synergistic effect between the parental genomes. The occurrence of characteristic peaks in venoms of hybrid populations might be used to identify these populations as well as to distinguish them from their parents.
Apis mellifera; venom; hybrid populations; molecular exclusion chromatography; honeybees
MOLECULAR EXCLUSION CHROMATOGRAPHY OF CRUDE VENOM AS AN AUXILIARY TOOL TO IDENTIFY HYBRID HONEYBEE POPULATIONS.
M.S. PALMA
, M.R. BROCHETTO-BRAGA
, J. CHAUD-NETTO , O. MALASPINA , M.R. OLIVEIRA
.
1 Department of Biology, Institute of Biosciences of Rio Claro, State of São Paulo, Brazil, 2 Center for the Study of Social Insects - CEIS - UNESP, State of São Paulo, Brazil, 3 Center for the Study of Venoms and Venomous Animals - CEVAP - UNESP, State of São Paulo, Brazil.
ABSTRACT. A comparison among the profiles of molecular exclusion chromatography in Sephadex G 100 column of venoms from Apis mellifera adansonii and Africanized honeybees revealed unique peaks which might be used to identify these populations. The venoms from hybrid populations resulting from the reciprocal mating of Apis mellifera adansonii and Africanized honeybees presented unique peaks, probably resulting from a synergistic effect between the parental genomes. The occurrence of characteristic peaks in venoms of hybrid populations might be used to identify these populations as well as to distinguish them from their parents.
KEY WORDS:Apis mellifera; venom; hybrid populations; molecular exclusion chromatography; honeybees.
INTRODUCTION
Efforts have been made to develop methods to allow the detection of genotypes which might be used to characterize specific races of honeybees. In addition to morphometrical analysis(1,8) the approaches currently used include: enzyme polymorphisms(9), DNA restriction fragment polymorphisms(2,3), analysis of hydrocarbons both from cuticula(5) and sting apparatus(6).
By using molecular exclusion chromatography, Palma & Brochetto-Braga in 1993(7) demonstrated some differences in venom composition from Apis mellifera ligustica, Apis mellifera adansonii and Africanized honeybees, which could be used to identify each race. We decided to investigate if this procedure might be used to follow some molecular markers from parentals to their progeny. Thus, venoms from Apis mellifera adansonii, Africanized honeybees and their descendants obtained by reciprocal matings of these races were fractionated.
MATERIAL AND METHODS
Colonies of Africanized honeybees and Apis mellifera adansonii were maintained in the apiary of the Institute of Biosciences at the São Paulo State University, Campus of Rio Claro, Brazil.
The colonies of Apis mellifera adansonii were descendants from queens originally brought from Ghana, Africa.
To ensure the genetic homogeneity of each race, all colonies used in this study were maintained through instrumental insemination, using semen from a single drone of the same maternal race in each crossing. Hybrid colonies of the Africanized and Apis mellifera adansonii populations were also produced by instrumental insemination.
The age of the honeybee workers was determined by marking the thorax of newly emerged individuals in order to follow them. Honeybee workers aged between 25 and 30 days were captured with an insect net, near the entrance of the colonies of each race, on the same day. The captured bees were immediately cooled on ice, frozen and stored at -16ºC until dissection.
The venom was obtained as described by Palma & Brochetto-Braga(7). Protein concentrations were determined by the method of Lowry et al.(4) using bovine serum albumin as standard.
The venoms (13 mg/ml) were placed on a Sephadex G-100 column (52x2cm), previously equilibrated with 10mM of ammonium acetate at pH 6.8. The elutions were carried out at 10ml/hr, collecting fractions of 3ml. The column eluent was monitored by protein spectrophotometric determination.
The native molecular weight of each eluted fraction was estimated using the molecular exclusion column in the same conditions described above. The column of Sephadex G-100 was previously calibrated with the following molecular weight (MW) markers: bovine serum albumin (MW 67kD); ovalbumin (MW 43kDa); trypsin (MW 22kDa); lisozyme (MW 16.6 kDa) and aprotinin (MW 6.5kDa).
RESULTS AND DISCUSSION
The chromatography profiles revealed 29 different peaks with MW from 7.5 to more than 100 kDa as observed in Figures 1, 2, 3, 4 and Table 1.
Occurrence and percentual concentration of each fraction of protein obtained after molecular exclusion chromatography in Sephadex G-100 column.
A comparison between the profiles of venom fractionation from Apis mellifera adansonii and Africanized honeybees showed great differences which could be used to distinguish them.
The venom of Apis mellifera adansonii (Figure 3) presented 19 peaks; 14 of them being characteristic of this race: 9, 14, 15, 17, 18, 19, 20, 21, 22, 24, 26, 27, 28 and 29.
The venom from Africanized honeybees (Figure 4) presented 8 peaks, three of them were unique, namely: peaks 1, 2 and 3 (MW > 100 kDa). Thus, the venom from Apis mellifera adansonii is characterized by a great number of protein peaks with MW from 7.5 to 38 kDa, while the Africanized bee venom is characterized both by a small number of protein peaks in the same region of MW and by three peaks of high MW. Table 1 shows that the venoms from African and Africanized honeybees have five common peaks: 5, 7, 13, 16 and 23, four of them detected in higher percentages in venom from Africanized honeybees than in venom of African honeybees. In addition to this, Table 1 shows that the proteins with MW higher than 7.5 kDa correspond to 64.3 and 42.7% of the total venoms from Africanized honeybees and Apis mellifera adansonii, respectively.
The mating of an Apis mellifera adansonii queen with an Africanized drone produced descendants which presented 15 peaks of proteins with MW from 13.5 to 87 kDa (Figure 1); 9 of these peaks (nº. 5, 7, 9, 13, 14, 15, 17, 19 and 22) were common to the female ascendancy (Apis mellifera adansonii), including concerning the protein concentrations (Table 1); three peaks (nº. 5, 7 and 13) were common to both parentals and five peaks were characteristic of their offspring (nº. 8, 10, 11, 12 and 25), as they were absent in the parental venoms.
The instrumental insemination of an Africanized queen using sperms of an Apis mellifera adansonii drone produced descendants which presented twelve peaks of proteins (Figure 2) with MW from 12 to more than 100 kDa; three of these peaks (nº 1, 2 and 3) were common to the female ascendancy (Africanized honeybees); one peak was common to male ascendancy (nº 20); four peaks (nº. 5, 13, 16 and 23) were common to both parentals; one peak (nº 4) was characteristic of these descendants and three peaks (nº 8, 10 and 25) were common to descendants of the anterior reciprocal crossing and may be considered as typical of hybrid populations.
The appearance of new protein peaks, unique for the F1 of intermated Apis mellifera adansonii and Africanized parentals, seems to indicate a probable synergistic effect. Thus, the molecular exclusion chromatography of venoms in Sephadex G-100 columns, revealed to be a powerful supporting tool for chemotaxonomic purposes to distinguish different honeybee populations, as we found several phenotypic protein peaks characteristic of each progeny, which might be used as molecular markers for genotypes of Apis mellifera adansonii, Africanized honeybees and the bees resulting from the reciprocal matings of parentals from both populations.
References
- 01 DALY HV., BALLING SV. Identification of Africanized honeybees in western hemisphere by discriminant analysis. J. KansasEntomol. Soc., 1978, 51, 145-87.
- 02 HALL G. DNA differences found between Africanized and European honeybees. Proc. Natl. Acad. Sci., 1986, 83, 4874-7.
- 03 HALL G. Distinguishing African and European honeybees using nuclear DNA restriction fragment polymorphisms. J. Apic. Res., 1988, 21, 93-7.
- 04 LOWRY OH., ROSEMBROUGH NJ., FARR AL., RANDALL RJ. Protein measurement with the folin phenol reagent. J. Biol. Chem., 1951, 193, 265.
- 05 McDANIEL CA., HOWARD RW., BLOMQUIST GJ., COLLINS A. Hydrocarbons of the cuticle, sting apparatus and sting shaft of Apis mellifera L. identification and preliminary evaluation as chemotaxonomic characters. Sociobiol., 1984, 8, 287-98.
- 06 McDANIEL CA., HOWARD RW., COLLINS AM., BROWNS WA. Variation in the hydrocarbon composition of non-Africanized Apis mellifera L. sting apparatus. Sociobiol., 1987, 13, 133-43.
- 07 PALMA MS., BROCHETTO-BRAGA MR. Biochemical variability between venoms from different honeybee (Apis mellifera) races. Comp. Biochem. Physiol., 1993, 106C, 423-7.
- 08 RINDERER TE. Computer assisted identification of hybrid strains of western honeybees. Flor. Entomol., 1988, 71, 281-8.
- 09 SYLVESTER HA. Electrophoretic identification of Africanized honeybees. J. Apic. Res., 1982, 21, 93-7.
CORRESPONDENCE TO:Publication Dates
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Publication in this collection
11 Jan 1999 -
Date of issue
1995
