MATERIALS AND METHODS

Preparation of Samples

Collection of Samples
Stingless bees, Tetragonula spp., were collected from Alaminos, Laguna and
Tayabas, Quezon. A total of 80 stingless bees were obtained - 40 stingless bees from each
of the two localities. The samples were placed in separate tubes, labeled and stored in a
-20 C freezer to preserve the enzymes to be studied.

Crude Extract Preparation

Ten of the bees from each locality were individually placed in a ceramic mortar
containing 150 l of homogenizing buffer (0.01 ml 2-mercaptoethanol: 50 ml gel buffer)
and macerated using a micro pestles. This was done on ice to prevent the denaturation of
enzymes. After extraction, the crude enzyme extracts were subjected to starch gel
electrophoresis.

18

Electrophoresis

Preparation of Starch Gel

The starch gels were prepared a day before the actual electrophoretic run. Twelve
percent (12%) potato starch gels were prepared by dissolving 5.76 g potato starch in 48
mL dilute Tris-His buffer at pH 8. The Tris-His buffer was prepared by combining 50 ml
0.009 M Tris with 0.005 M Histidine buffer and 950 ml water (Appendix A). The mixture
was transferred in a 250-mL Erlenmeyer flask and was boiled in a microwave with
constant stirring every 5 seconds. This was done until the mixture became clear and
homogenous. The gels were then deaerated using a vacuum pump to remove the bubbles.
The mixture was poured in molders that were coated with glycerol that would prevent the
gel from sticking into the molder. A comb, also coated with glycerol, was placed on each
gel to make wells. The gels were then allowed to cool in a room temperature and were
later stored inside the refrigerator.

Loading of Samples
The gels were taken out the refrigerator and their combs were removed after the
gels solidified. Using a piece of filter paper, moistures from the wells were removed to
prevent the dilution of the crude extract. Using a micropipettor, each of the first 10 wells
was filled with 60 L of the crude extract while the 11 th and 12th wells were filled with 60

19

L of pooled sample and tracking dye (1.004% bromphenol blue), respectively. The
pooled sample of each of the stingless bee population was prepared by obtaining 10 l
each of the previous samples each then mixing them into a 1.5 mL tube.

Electrophoresis Run
The gels were then mounted on the electrode trays and were run using Bio-Rad
gel electrophoresis, which can run three gels at the same time. Cold running buffer, TrisHis buffer at pH 8 (Appendix A), was poured into the troughs of the set-up until all gels
are completely submerged. The set-up was covered and was set to run from negative to
positive with 70 volts as the initial voltage. The voltage was then increased to 100 volts
after 30 minutes. The run was stopped when the tracking dye has reached the bottom of
the gel.

Slicing, Staining, and Fixation

After the run, the set-up was disassembled and the gels were carefully removed
from the glass plates. The gels were then transferred into an acrylic-slicing bed. A
diagonal cut was made on the upper corner of the gel, side opposite to the wells, where
the tracking dye was located to serve as the marker. The gels were sliced horizontally into
four 2 mm thin gels using a wire fastened to a copping saw. Then the gels were placed in

20

separate containers each containing a particular enzyme staining solution (Appendix B).
The gels were incubated in the dark until bands appeared.
After the stains were removed, the gels were washed with destaining solution (1
acetic acid: 5 methanol: 5 distilled water) for five seconds (Appendix A). The solution
was removed and was replaced with distilled water. The water was then drained and the
gels were covered with cling wrap and were stored in the refrigerator until ready for
scoring of bands.

Analysis of Data

Scoring of Bands
To visualize the bands better, the gels were viewed using a light box. The bands
seen on the gels were marked on an acetate. The distances of the marked bands were
measured including the distance traveled by the tracking dye. The measured values were
then used to compute for the relative mobilities of the bands, using the formula:

Rf =

Distance traveled by the proteinband

Distance tr aveled by the tracking dye

21

An electrophoretogram was constructed for each gel based on the computed Rf

values. The zones of enzymatic activity were recorded with the right orders noted based
on the increasing anodal migration. The loci were numbered from the slowest migrating
band to the fastest. Possible loci and genotypes were obtained from the banding pattern of
an enzyme. For each pattern, a corresponding genotype was designated composed of two
letters per locus which represents two alleles. Alleles were designated as slow (S) or fast
(F) according to their electrophoretic mobilities.

Determination of Genetic Variation

From the bands found in each locus, the number of loci and of the alleles were
determined for each enzyme system. The genotype of each sample, and the genotype and
gene frequencies were then computed and were used in estimating genetic variation
within and among the population of stingless bees from Laguna and Quezon.

Measure of Intrapopulation Variation

The intrapopulation variation was estimated by determining the proportion of
polymorphic loci (P), average number of alleles per locus (A), and mean heterozygosity
(He). The variable P expresses the percentage of variable loci in a population. This was
assessed by first determining if the locus is polymorphic or not. Polymorphism is
ascertained if the frequency of the most common allele (q) is at most 0.95 and at least

22

0.05 (0.05 < q < 0.95), otherwise the loci was deemed monomorphic (Nei, 1975). The
variable P was then computed using the formula shown below:

P=

Number of polymorphic locieach population

Totalnumber of loci

The variable A was used to determine the number of allelic variants present in a
gene, which was computed using the formula shown below (Nei, 1975):

A=

Total number of alleles present all theloci examined

Totalnumber of loci present

The mean heterozygosity (He), which was used to measure the extent of genetic
variability in the population of stingless bees, was obtained by computing for the
summation of the average heterozygosity of each locus by the total number of loci. The
formula for computing the average heterozygosity is shown below:
H=1 X 2i

Where:

23

Xi = frequency of the ith allele at a locus.

Measure of Interpopulation Variation
The interpopulation was measured by computing for the genetic identity (I N),
genetic distance (D), and the genotypic similarity or genotypic identity (I). The variable
IN, which is the degree of similarity between different populations, was determined by
using gene frequencies of randomly mating diploid populations labeled as x and y. The
normalized identity of genes between x and y with respect to each locus (I j) was
calculated with the formula shown below

I j=

j xy
jx j y

Where:
n

jx =

x 21
i=1

is the probability of identity of two randomly chosen genes in

population x;
n

jy =
population y;

y 21
i=1

is the probability of identity of two randomly chosen genes in

24
n

jxy =

x 1 y 21
i=1

is the probability of identity of a gene from population x and a

gene from population y and ;

n = number of genes at locus j.

When the two populations have the same alleles in equal frequencies, Ij is
equivalent to unity or one (1), but become zero (0) when no alleles are common to the
two populations.
The genetic identity between x and y with respect to all loci (I) was computed
with the formula shown below:

I=

J xy
( J x J y )

Where:
Jxy, Jx, Jy are the means of jxy, jx, and jy of all loci, respectively.

25

The genetic distance (D), which measures the degree of difference between the
populations, was computed on the assumption that the rate of gene substitution per locus
was the same for all loci using the formula shown below:

D=ln ( I N )

Hedricks probability of genotypic identity (I) was then used to compare

genotypic frequencies of populations. It was computed using the following equation:

p + p 2i y
2
ix

i =1
n

i=1

pi x pi y

I x y = i=1

Where:
Pjx = frequency of the jth genotype in population x;
Pjy = frequency of the jth genotype in population y;

26

n = number of genotypes at the locus;

numerator = the probability of drawing identical genotypes from the two
populations and;
denominator = the average probability of drawing identical genotypes from the
same population on two successive independent draws.