**3.4 MW determination**

358 Gene Duplication

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* 

Table 2 shows the results obtained for the esterase activity patterns of late third instar larvae of the analyzed species in the presence of different inhibitors. All species showed the same pattern for EST-4: they were inhibited only by PMSF. For EST-5, all species were inhibited

Species Enzyme PMSF Malathion CuSO4 IAC E-64 Eserine pCMB HgCl2 *D. mulleri* EST-4 ++ ⊗ ⊗ ⊗⊗ ⊗ ⊗ ⊗

*D. aldrichi* EST-4 ++ ⊗ ⊗ ⊗⊗ ⊗ ⊗ ⊗

*D. wheeleri* EST-4 ++ ⊗ ⊗ ⊗⊗ ⊗ ⊗ ⊗

*D. mojavensis* EST-4 ++ ⊗ ⊗ ⊗⊗ ⊗ ⊗ ⊗

*D. arizonae* EST-4 ++ ⊗ ⊗ ⊗⊗ ⊗ ⊗ ⊗

*D. navojoa* EST-4 ++ ⊗ ⊗ ⊗⊗ ⊗ ⊗ ⊗

Table 2. Esterase activity patterns of EST-4 and EST-5 for the six *Drosophila* species analyzed in the presence of different inhibitors. PMSF = phenylmethylsulfonyl fluoride; Eserine = eserine sulfate; CuSO4 = copper sulfate; IAC = iodoacetamide; E-64 = trans-epoxysuccinyl-Lleucyl-amido(4-guanidino) butane; pCMB = p-chloromercuribenzoate; HgCl2 = mercuric

The I.P. determination was performed in two phases. In the first phase, we verified that the best range for I.P. determination was 6.0 to 8.0. In the second phase, an ampholyte solution was used for this pH range. Table 3 shows that all esterases presented I.P. between 6.0 and 7.0. As expected, the I.P. values for EST-5 in both larvae and adults of the same species were equal, ranging from 6.47 (*D. navojoa*) to 6.64 (*D. aldrichi*). EST-4 showed a wider range of I.P. variation than EST-5, with *D. mulleri* and *D. navojoa* showing the highest and lowest I.P.

EST-5 ⊗ ++ ⊗ ⊗⊗ ⊗ ⊗ ⊗

EST-5 ⊗ ++ ⊗ ⊗⊗ ⊗ ⊗ ⊗

EST-5 ⊗ ++ ⊗ ⊗⊗ ⊗ ⊗ ⊗

EST-5 ⊗ ++ ⊗ ⊗⊗ ⊗ ⊗ ⊗

EST-5 ⊗ ++ ⊗ ⊗⊗ ⊗ ⊗ ⊗

EST-5 ⊗ ++ ⊗ ⊗⊗ ⊗ ⊗ ⊗

**3.2 Pattern of esterase activity in the presence of inhibitors** 

chloride. ++ activity inhibited; ⊗ activity not affected.

**3.3 Isoelectric point (I.P.) determination** 

values (6.88 and 6.37, respectively).

only by malathion. No other inhibitor affected the activity of either esterase.

(data not shown).

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

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 weights are presented in Table 4.

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.

Gene Duplication and Subsequent

and EST-5 are dimeric in both species.

**3.5.1 Crosses between** *D. mulleri* **and** *D. mojavensis*

**3.5.2 Crosses between** *D. navojoa* **and** *D. mojavensis*

*mulleri*; 2 = *D. mojavensis*; 3-13 = hybrid larvae.

cross between *D. navojoa* females and *D. mojavensis* males was very fertile.

**3.5 Interspecies crosses** 

these species.

13; Figure 5).

Differentiation of Esterases in Cactophilic *Drosophila* Species 361

The cross between *D. mulleri* and *D. mojavensis* showed asymmetric isolation, with many descendents only in the direction of *D. mulleri* females and *D. mojavensis* males. The reciprocal cross did not produce offspring despite the presence of courtship among couples and eggs in the plate. The hybrid larvae were analyzed in 10% PAGE and showed threeband patterns for EST-4 and EST-5 (Figure 4). For both enzymes, the slower band corresponded to the enzyme encoded by *D. mulleri* and the faster to the enzyme encoded by *D. mojavensis*. The intermediate band represented a hybrid enzyme, indicating that EST-4

For EST-4, the hybrid intermediate bands were located closer to the band encoded by *D. mulleri*, which could be a result of differences in the I.P. values of theses enzymes (Table 3). The same was not observed for EST-5, as this enzyme has nearly the same value for both of

Asymmetric isolation was also observed in the cross between *D. navojoa* and *D. mojavensis.*  No offspring were obtained in the direction of *D. mojavensis* females and *D. navojoa* males, despite the fact that courtship between couples and eggs on the plate were observed. The

Fig. 4. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines and the hybrids obtained from the cross of *D. mulleri* females and *D. mojavensis* males. 1 = *D.* 

The hybrid larvae from this cross were analyzed in 10% PAGE and showed the same threeband patterns observed for *D. mulleri* and *D. mojavensis* (Figure 5). For EST-4, the slower band corresponded to the enzyme encoded by *D. mojavensis*, the faster band corresponded to the enzyme encoded by *D. navojoa*, and the intermediate band corresponded to a hybrid enzyme. The opposite was observed for EST-5: the slower band was from *D. navojoa*, the faster band was from *D. mojavensis*, and an intermediate band was a hybrid enzyme. Again, these results confirm the quaternary structure of both enzymes of these species. An interesting observation was the absence of EST-5 expression in two samples (samples 12 and

Fig. 3. Log of Rm versus gel concentrations relationship (Ferguson's plot) for EST-4 and EST-5. A – *Drosophila mulleri*; B – *Drosophila aldrichi*; C – *Drosophila wheeleri*; D – *Drosophila navojoa*.


Table 4. Slopes of Log Rm versus gel concentration relationships and MW estimates for EST-4 and EST-5 of the six cactophilic *Drosophila* species analyzed.1 Data obtained from Mateus et al. (2009).
