3.3.1.1. Brief description of procedures

We here give a very brief overview over the steps involved. 

Extraction:
a. Dissect the bee and place thorax in a microtiter plate well.
b. Grind the thorax with 20 µl of Tris-HCl buffer (for MDH, ME, EST, PGM and HK, see table 5). Use the whole body for the study of ALP (see table 6).

Electrophoresis: good results can be obtained with the use of 7.5% polyacrylamide gel (Davis, 1964) with Tris-Glycine buffer pH 8.3.
Caution: acrylamide in unpolymerized form is a potent neurotoxin! Observe operator safety when handling (wear gloves, work under fume hood)! It is recommendable to cast the gels using acrylamide solution and we discourage the use of acrylamide powder. The liquid preparation is much easier to handle and presents less of a health hazard. A good alternative to polyacrylamide alternative is the purchase and use of ready-cast cellulose acetate gels.

 Staining:
a. prepare locus-specific stain solution according to recommendations.
Recommendations for substrate systems, stains and buffers have been published in: Boyer, 1961; Gahne, 1967; Shaw and Prasad, 1970; Harris and Hopkinson, 1976. We have included recommendations for stains in Table 6.
Obey safety precautions, since many ingredients may pose a health hazard!
b. immerse gel into solution,
c. incubate in the dark according to recommendations (specific for different loci, no general recommendation can be given here).

Scoring: It is the convention to score the most common band as locus100 and measure the mobility of all other bands in relation to this band (e.g., Mdh100, Mdh65).

Table 6. Most polymorphic and most commonly used enzyme loci for allozyme studies. E.C. Enzyme commission number of the locus (www.chem.qmul.ac.uk/iubmb/enzyme).

Polymorphic locus

Known Alleles

References

Staining recommendation

MDH-1

 

EC 1.1.1.37

 

MDH65 MDH80 MDH87 MDH100

MDH116

MDH125

MDH133

Garstide, 1980; Nunamaker et al., 1984; Badino et al., 1983, 1985, 1988; Sheppard, 1988; Sheppard and Berlocher, 1984, 1985; Sheppard and McPheron, 1986; Lobo et al., 1989; Meixner et al., 1994; Kandemir and Kence, 1995; Smith and Glenn, 1995; Dedej et al., 1996; Kandemir et al., 2000; Bouga et al., 2005; Ivanova, 2010; Ivanova et al., 2007, 2011

NAD (0.035 g)

NBT (0.03 g)

PMS (0.0015 g)

Na malate (10 ml)

Phosphate buffer (pH 7.3) – 100 ml

ME

 

EC 1.1.1.40

 

 

ME70

ME100

ME106

ME117

 

Sheppard and Berlocher, 1984, 1985; Sheppard and McPheron, 1986; Meixner et al., 1994; Dedej et al., 1996; Kandemir et al., 2000, 2005; Bouga et al., 2005; Ivanova, 2010, Ivanova et al., 2011

NADP (0.035 g)

NBT (0.03 g)

PMS (0.0015 g)

Na malate (10 ml)

Phosphate buffer (pH 7.3) – 100 ml

EST 3

 

EC 3.1.1

 

 

EST70

EST80

EST88

EST94

EST100

EST105

EST118

EST130

Sheppard and McPheron, 1986; Kandemir and Kence, 1995; Bouga et al., 2005; Ivanova, 2010; Ivanova et al., 2010a, b, c, 2011

 

 

1-naphtil acetate Na salt (0.010 g)

2-naphtil acetate Na salt (0.020 g)

Fast blue BB salt (0.050 g)

Phosphate buffer (pH 7.0) – 100 ml

ALP

 

EC 3.1.3.1

ALP80

ALP90

ALP100

Bouga et al., 2005; Ivanova, 2010; Ivanova et al., 2010a, b, c; Ivanova et al., 2011

 

1-naphtil phosphate Na salt (0.020 g)

Fast garnet (0.050 g)

MgCl2 (0.120 g)

0.1 M TRIS-HCl buffer (pH 8.6) – 100 ml

PGM

 

EC 5.4.2.2

 

 

 

 

 

PGM75

PGM80

PGM100

PGM114

PGM120

 

 

 

Mestriner and Contel, 1972; Brueckner, 1974; Nunamaker and Wilson, 1980; Badino et al., 1983; Sheppard and Berlocher, 1985; Del Lama et al., 1985; Meixner et al., 1994; Smith and Glenn, 1995; Kandemir and Kence, 1995; Ivanova, 2010; Ivanova et al., 2010a, b, c; Ivanova et al., 2011

G-1-Phosphate Na salt (0.6 g)

MgCl2.6H2O (0.2 g);

NADP (0.01 g)

NBT (0.020 g)

PMS (0.001 g)

G-6-P-D (80 U (0.035 mg))

0.5 M TRIS-HCl buffer (pH 7.1) – 10 ml

H2O – 90 ml

HK

 

EC 2.7.1.1

 

 

 

HK77

HK87

HK100

HK110

HK120

 

 

Sheppard and McPheron, 1986; Badino et al., 1988; Del Lama et al., 1988, 1990; Smith and Glenn, 1995; Kandemir and Kence, 1995; Kandemir et al., 2000; Ivanova et al., 2010a, b, c; Ivanova et al., 2011

Glucose (0.09 g)

MgCl2.6H2O (0.02 g)

ATP (0.025 g)

NADP (0.025 g)

NBT (0.020 g)

PMS (0.003 g)

G-6-P-D (80 un (0.035 mg))

0.5 M TRIS-HCl buffer (pH 7.1) – 10 ml

H2O – 90 ml