**5. Conclusions**

366 Gene Duplication

In all of these crosses, the phenotypic observations of the esterase patterns from late third instar hybrid larvae produced three bands for both EST-4 and EST-5 (Figures 4, 5, 6 and 7), except for larvae from the cross between *D. navojoa* and *D. arizonae,* which produced a thicker band because the parental bands have almost the same migration pattern under the electrophoretic conditions used in this study. These results indicate that in all six *Drosophila*  species, EST-4 and EST-5 have dimeric quaternary structures. Another important observation from some of these crosses was the presence of hybrid larvae with no EST-5 activity (*D. navojoa* x *D. mojavensis* – Figure 5; *D. navojoa* x *D. arizonae* – data not shown). These results indicate that some hybrid larvae had problems with the regulation of *Est-2a* gene expression, which most likely codes for the EST-5 enzyme, without affecting the expression of its homologous gene, *Est-2c*, which most likely codes for the EST-4 enzyme (Robin et al., 2009). These results reinforce the idea that these two loci are independent. The possible role of EST-4 in these *Drosophila* species remains an open question. According to Holmes & Masters (1967, as cited in Oakeshott et al., 1993), esterases can be classified into four types through their specific inhibition patterns. Carboxylesterases are esterases that are inhibited only by organophosphates, such as paraoxon, fenitrooxon and DFP (diisopropylfluorophosphate). Cholinesterases are inhibited by organophosphates and carbamates, such as eserine sulfate. Arylesterases are inhibited only by sulfhydrylic agents, such as p-chloromercuribenzoate (pCMB). Acetylesterases are not inhibited by any of these agents. Inhibition of EST-5 only by malathion, an organophosphate, suggests that this enzyme belongs to the class of carboxylesterases. Inhibition of EST-4 by PMSF and the absence of inhibition in the presence of all other inhibitors tested suggest that this enzyme

According to Augustinsson (1968), esterases are closely related to the class of serineproteases, forming a multigenic family of serine-hydrolases. The main features that support this hypothesis are the three consensus amino acid residues that are present in the active site of esterases and serine-proteases, including an invariant serine, enzymatic inactivation by DFP, which binds irreversibly to the serine residue of both enzymes, inhibition by organophosphates and carbamates and the superposition of substrate preference (Augusteyn et al., 1969; Krisch, 1971; Dayhoff et al., 1972; Heymann, 1980; Previero et al., 1983; as cited in Myers et al., 1988). However, Myers et al. (1988) showed that some esterases cannot be included in this multigenic family because they do not have the same amino acid

The absence of EST-4 and Est-5 inhibition by copper sulfate and iodoacetamide, combined with data for E-64, which is a diagnostic inhibitor of cysteine-proteases, indicate that neither enzyme has an essential cysteine residue in its active site. On the other hand, the inhibition of EST-4 by PMSF, which is a diagnostic compound for serine-proteases and other enzymes with a serine residue in the active site, and of EST-5 only by malathion indicated that both enzymes have an important serine residue in the active site, suggesting that they belong to the class of serine-hydrolases. As these enzymes display high esterase activity, we can postulate that they are serine-esterases (Holmes & Masters, 1967). The multigenic family of serine-esterases includes several enzymes with a wide range of functions, including cholinesterases, lipases, lysophospholipases, cholesterol-esterases, non-specific carboxylesterases and juvenile hormone esterases (Ryger et al., 1989; Doctor et al., 1990; Shimada et al., 1990; as cited in Myers et al., 1993). Therefore, EST-4 and EST-5 probably belong to this multigenic family, with EST-4 as an acetylesterase (E.C. 3.1.1.6) and EST-5 as a

probably belongs to the class of acetylesterases.

non-specific carboxylesterase (E.C. 3.1.1.1).

residues in the charge exchange system of the enzyme active site.

This study contributes to a better understanding of the differentiation of two enzymes that are products of a gene duplication in six cactophilic *Drosophila* species. We present additional evidence to support the gene duplication event that gave rise to the genes responsible for the EST-4 and EST-5 enzymes, which are the main β-esterases found in several species of the *D. mulleri* complex of the *D. repleta* group. We also contribute to the elucidation of the possible physiological roles of these esterases in this group. Further steps in this investigation will be to determine specific biochemical parameters of both enzymes after purification. We are also interested in identifying the changes that occur in the regulatory system of gene expression that lead to differentiation in the patterns of tissuespecific and temporal expression of these enzymes; that is, understanding what triggers EST-4 expression only in the late third instar larvae and at the larval carcass. We are also interested in determining the intra- and/or extracellular processes in which these enzymes are involved and their interacting molecules. Thus, we will be able to complement this initial step with an increased understanding of the differentiation of these two genes that result from a gene duplication event.
