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

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


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

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

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. values (6.88 and 6.37, respectively).


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.
