**2.4 Esterase detection**

Esterase detection was performed using 10% polyacrylamide gel electrophoresis (PAGE), adapted by Ceron (1988) from Davis (1964) and Laemmli (1970). All samples were prepared in 25 µL of 0.1 M Tris-HCl (pH 8.8) buffer containing 10% glycerol, where 10 µL was used in the gels. After electrophoresis, all gels were soaked in 0.1 M phosphate buffer (pH 6.2) for 1 hour at 25ºC. After this period, the gels were stained in solution containing 100 mL of phosphate buffer, 10 ml of n-propanol and 120 mg of Fast Blue RR Salt, where 40 mg of αnaphthyl acetate and 30 mg of β-naphthyl acetate, previously dissolved in 2 ml of acetone, were added. After approximately 1 hour, the staining reactions were stopped in a solution of acetic acid:ethanol:water (1:2.5:6.5 by v:v:v). Because the esterases hydrolyze substrates

Gene Duplication and Subsequent

Arrow = EST-4; arrowhead = EST-5.

arrowhead = EST-4; smaller arrowhead = EST-5.

Differentiation of Esterases in Cactophilic *Drosophila* Species 357

Fig. 1. Esterase pattern in 10% PAGE for late third instar larvae and adult females of *Drosophila mulleri* (1 and 2 = larvae; 3 = female), *D. arizonae* (4 and 5 = larvae; 6 = female), *D. mojavensis* (7 and 8 = larvae; 9 = female), *D. navojoa* (10 and 11 = larvae; 12 = female), *D. wheeleri* (13 and 14 = larvae; 15 = female), *D. aldrichi* (16 to 19 = larvae; 20 = female). All wells contain individual samples, except for wells 18 and 19, which contain 2 larvae of *D. aldrichi*.

Fig. 2. A. 10% PAGE showing the electrophoretic staining differences for EST-4 and EST-5. 1- *D. mojavensis*; 2 - *D. arizonae*; 3 - *D. navojoa*; 4 - *D. mulleri*; 5 - *D. aldrichi*; 6 - *D. wheeleri*. B. 10% PAGE of late third instar larvae of *D. navojoa*, showing the different phenotypes observed. Arrows in A and B indicate the interlocus heterodimer EST-4/EST-5. Larger

differently, the bands in the gel stain differently: black when they hydrolyze α-naphthyl acetate, red when they hydrolyze β-naphthyl acetate and magenta when they hydrolyze both α- and β-naphthyl acetates. Polyacrylamide gels were air dried at room temperature using gelatin and cellophane wound slab gels in an embroidering hoop (Ceron et al., 1992).

### **2.5 Characterization of esterases using inhibitors**

Malathion, phenylmethylsulfonyl fluoride (PMSF), eserine sulfate, copper sulfate (CuSO4), iodoacetamide (IAC), trans-epoxysuccinyl-L-leucyl-amido(4-guanidino) butane (E-64), pchloromercuribenzoate (pCMB) and mercuric chloride (HgCl2) were used as specific inhibitors, all in 1 mM concentrations (with the exception of E-64, which was used at a concentration of 5 mM) in the soaking and staining solution.

### **2.6 Determination of isoelectric point (I.P.)**

The I.P. was determined in 10% PAGE containing 5% ampholyte solution (Sigma). The first ampholyte formed a pH gradient between 3.0 and 10.0 after 1 hour of constant 100 V prefocusing. This experiment was performed to verify the best gradient to determine the I.P. values of all enzymes in all species. After this verification, another ampholyte was used that formed a pH gradient between 6.0 and 8.0 after 1 hour of constant 100 V pre-focusing. In both cases, ampholyte solutions were added before gel polymerization. Samples of the six *Drosophila* species and of the I.P. marker (hemoglobin) were prepared in a 10% glycerol in water solution. Esterase isoelectric focusing was performed under constant 100 V conditions in the power supply for 3 hours. After focusing, the gels were soaked in buffer for 1 hour, followed by esterase staining for the same period, as described in section 2.5. Following esterase identification, the gels were stained for total protein with Coomassie Blue G250 overnight. The staining reaction was stopped, and the gels were dried as described in section 2.5. The I.P. was estimated by comparing the positions of EST-4 and EST-5 with the position of human hemoglobin (I.P. = 7.1) after focusing.

### **2.7 Molecular weight (MW) estimation**

The MW estimation was performed using the method adapted by Mateus et al. (2009) from Hedrick and Smith (1968). The following standard MW proteins were used: myoglobin (17.8 kD), soybean trypsin inhibitor (24 kD), carbonic anhydrase (29 kD), ovalbumin (45 kD), human serum albumin (66 kD) and phosphorylase-b (97.4 kD). All graphics were constructed using Microcal Origin software, version 3.5 (Scientific and Technical Graphics in Windows – copyright 1991 – 1994, Microcal Software Inc.).

### **3. Results**

### **3.1 Esterase pattern**

Figure 1 shows the esterase patterns of larvae and adults (females) from six *Drosophila*  species. For all species, EST-4 always migrated slower than EST-5. The *D. navojoa* stock was the only one that had more than one band for EST-4. EST-4 was more strongly stained than EST-5 in *D. mulleri*, *D. aldrichi* and *D. wheeleri* (Figure 2A and B). The opposite was observed for *D. arizonae*, with EST-5 more strongly stained than EST-4 (Figure 2A). Differences in the staining intensity among EST-4 bands were also observed, with the *D. mojavensis* cluster species (*D. mojavensis*, *D. arizonae* and *D. navojoa*) showing fainter bands than the species of the *D. mulleri* cluster (*D. mulleri*, *D. aldrichi* and *D. wheeleri*).

differently, the bands in the gel stain differently: black when they hydrolyze α-naphthyl acetate, red when they hydrolyze β-naphthyl acetate and magenta when they hydrolyze both α- and β-naphthyl acetates. Polyacrylamide gels were air dried at room temperature using gelatin and cellophane wound slab gels in an embroidering hoop (Ceron et al., 1992).

Malathion, phenylmethylsulfonyl fluoride (PMSF), eserine sulfate, copper sulfate (CuSO4), iodoacetamide (IAC), trans-epoxysuccinyl-L-leucyl-amido(4-guanidino) butane (E-64), pchloromercuribenzoate (pCMB) and mercuric chloride (HgCl2) were used as specific inhibitors, all in 1 mM concentrations (with the exception of E-64, which was used at a

The I.P. was determined in 10% PAGE containing 5% ampholyte solution (Sigma). The first ampholyte formed a pH gradient between 3.0 and 10.0 after 1 hour of constant 100 V prefocusing. This experiment was performed to verify the best gradient to determine the I.P. values of all enzymes in all species. After this verification, another ampholyte was used that formed a pH gradient between 6.0 and 8.0 after 1 hour of constant 100 V pre-focusing. In both cases, ampholyte solutions were added before gel polymerization. Samples of the six *Drosophila* species and of the I.P. marker (hemoglobin) were prepared in a 10% glycerol in water solution. Esterase isoelectric focusing was performed under constant 100 V conditions in the power supply for 3 hours. After focusing, the gels were soaked in buffer for 1 hour, followed by esterase staining for the same period, as described in section 2.5. Following esterase identification, the gels were stained for total protein with Coomassie Blue G250 overnight. The staining reaction was stopped, and the gels were dried as described in section 2.5. The I.P. was estimated by comparing the positions of EST-4 and EST-5 with the

The MW estimation was performed using the method adapted by Mateus et al. (2009) from Hedrick and Smith (1968). The following standard MW proteins were used: myoglobin (17.8 kD), soybean trypsin inhibitor (24 kD), carbonic anhydrase (29 kD), ovalbumin (45 kD), human serum albumin (66 kD) and phosphorylase-b (97.4 kD). All graphics were constructed using Microcal Origin software, version 3.5 (Scientific and Technical Graphics in

Figure 1 shows the esterase patterns of larvae and adults (females) from six *Drosophila*  species. For all species, EST-4 always migrated slower than EST-5. The *D. navojoa* stock was the only one that had more than one band for EST-4. EST-4 was more strongly stained than EST-5 in *D. mulleri*, *D. aldrichi* and *D. wheeleri* (Figure 2A and B). The opposite was observed for *D. arizonae*, with EST-5 more strongly stained than EST-4 (Figure 2A). Differences in the staining intensity among EST-4 bands were also observed, with the *D. mojavensis* cluster species (*D. mojavensis*, *D. arizonae* and *D. navojoa*) showing fainter bands than the species of

**2.5 Characterization of esterases using inhibitors** 

**2.6 Determination of isoelectric point (I.P.)** 

concentration of 5 mM) in the soaking and staining solution.

position of human hemoglobin (I.P. = 7.1) after focusing.

Windows – copyright 1991 – 1994, Microcal Software Inc.).

the *D. mulleri* cluster (*D. mulleri*, *D. aldrichi* and *D. wheeleri*).

**2.7 Molecular weight (MW) estimation** 

**3. Results** 

**3.1 Esterase pattern** 

Fig. 1. Esterase pattern in 10% PAGE for late third instar larvae and adult females of *Drosophila mulleri* (1 and 2 = larvae; 3 = female), *D. arizonae* (4 and 5 = larvae; 6 = female), *D. mojavensis* (7 and 8 = larvae; 9 = female), *D. navojoa* (10 and 11 = larvae; 12 = female), *D. wheeleri* (13 and 14 = larvae; 15 = female), *D. aldrichi* (16 to 19 = larvae; 20 = female). All wells contain individual samples, except for wells 18 and 19, which contain 2 larvae of *D. aldrichi*. Arrow = EST-4; arrowhead = EST-5.

Fig. 2. A. 10% PAGE showing the electrophoretic staining differences for EST-4 and EST-5. 1- *D. mojavensis*; 2 - *D. arizonae*; 3 - *D. navojoa*; 4 - *D. mulleri*; 5 - *D. aldrichi*; 6 - *D. wheeleri*. B. 10% PAGE of late third instar larvae of *D. navojoa*, showing the different phenotypes observed. Arrows in A and B indicate the interlocus heterodimer EST-4/EST-5. Larger arrowhead = EST-4; smaller arrowhead = EST-5.

Gene Duplication and Subsequent

**3.4 MW determination** 

weights are presented in Table 4.

Differentiation of Esterases in Cactophilic *Drosophila* Species 359

*D. mulleri* **cluster I.P.** *D. mojavensis* **cluster I.P.** 

EST-4 6.88 EST-4 6.38

EST-5 (larvae and adult) 6.51 EST-5 (larvae and adult) 6.49

EST-4 6.55 EST-4 6.53

EST-5 (larvae and adult) 6.64 EST-5 (larvae and adult) 6.56

EST-4 6.59 EST-4 6.37

EST-5 (larvae and adult) 6.53 EST-5 (larvae and adult) 6.47

To determine the MW of both enzymes in all six *Drosophila* species analyzed, the technique described by Mateus et al. (2009) was applied using 6% to 12% PAGE and the same MW markers. The results presented there are part of this study. Therefore, in this study, we present the results that were not shown in Mateus et al. (2009), i.e., the MW determinations of EST-4 and EST-5 for *D. mulleri*, *D. aldrichi*, *D. wheeleri* and *D. navojoa*. After electrophoresis, the relative mobility (Rm) values for the esterases of these four species and the molecular markers were obtained. The graphs of Rm versus gel concentration for each MW marker resulted in a different slope. These slopes were plotted against the MW (Figure 1 – Mateus et al., 2009). Ferguson's plot (Log Rm versus gel concentrations) for EST-4 and

The plots for both esterases were parallel in all species, indicating that these enzymes have different charges and/or tridimensional structures but very similar molecular weights. From these graphs, the slope was obtained for each enzyme in each species. These values were used to estimate the MW in each case, using the equation *Y* = A + B*X*, where A is the intercept of the Y-axis (2.18766), and B is the slope (0.09452). The slopes and molecular

The slope values for both enzymes in all species were similar. EST-5 had more variation, ranging from -10.05407 ± 0.29546 for *D. navojoa* to -11.03429 ± 0.30178 for *D. mulleri*. EST-4 was less variable, ranging from -10.08361 ± 0.33581 for *D. wheeleri* to -10.52607 ± 0.44878 for *D. mulleri*. The MWs, estimated from these slope values (Table 4), were very close to each other. For EST-4, the MW ranged from 83.537 kD in *D. wheeleri* to 88.218 kD in *D. mulleri*. For EST-5, the MW ranged from 83.225 kD in *D. navojoa* to 93.595 kD in *D. mulleri*. The MWs

obtained, including standard deviations, were all approximately 80 kD to 96.8 kD.

Table 3. Isoelectric points for EST-4 and EST-5 of larvae and adults of the six analyzed *Drosophila* species, obtained through the comparison of esterase band mobility in gels with

an I.P. marker (hemoglobin; I.P. = 7.1) in a pH range between 6.0 and 8.0.

EST-5 of *D. mulleri*, *D. aldrichi*, *D. wheeleri* and *D. navojoa* are shown in Figure 3.

D. mulleri *D. mojavensis* 

*D. aldrichi D. arizonae* 

*D. wheeleri D. navojoa* 

Despite the observation of homozygotes for EST-4 in five out of six species analyzed, the quaternary structure for this enzyme as a dimer could be deduced from the presence of a heterodimer between EST-4 and EST-5 in *D. mojavensis* and *D. arizonae* (Figure 2A). This dimeric structure for EST-4 and EST-5 was confirmed by hybrid analyses. In *Drosophila navojoa*, in addition to the presence of the heterodimer, three phenotypes were observed in gels for EST-4 and EST-5 (Figure 2B): homozygous for a slower band (EST-4S and EST-5S, respectively); homozygous for a faster band (EST-4F and EST-5F, respectively); and heterozygous, with a three-band pattern. These patterns reinforce the quaternary structure of these enzymes for this species. The same results were observed for EST-5 of *D. mulleri*  (data not shown).
