**4. Discussion**

362 Gene Duplication

This cross was very fertile in both directions. However, larvae from the cross in the direction of *D. navojoa* females and *D. arizonae* males had very slow development and took much longer to achieve the late third instar stage; they also had a high mortality rate. The larvae analyzed in 10% PAGE from both cross directions presented the same three-band patterns for EST-5. For EST-4, as in both species of the cross, the enzymes had almost the same migration speed under these electrophoretic conditions. One thicker band was observed in the hybrid larvae, which must be the agglomeration of the three bands expected for this

Fig. 5. Esterase pattern in 10 % PAGE of late third instar larvae from the parental lines and the hybrids obtained from the cross of *D. navojoa* females and *D. mojavensis* males. 1 = *D.* 

Fig. 6. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines of *D. arizonae* and *D. navojoa* and the hybrids obtained from crosses in both directions. 1 = *D.* 

The cross between *D. navojoa* and *D. mulleri* was fertile in both directions. The larvae analyzed by 10% PAGE showed three-band patterns for both EST-4 and EST-5, with the slower enzyme from *D. mulleri* and the faster band from *D. navojoa.* The intermediate band was a hybrid enzyme. These results confirm the dimeric quaternary structure of these

**3.5.3 Crosses between** *D. navojoa* **and** *D. arizonae*

*navojoa*; 2 = *D. mojavensis*; 3-14 = hybrid larvae.

*arizonae*; 2 = *D. navojoa*; 3-15 = hybrid larvae.

**3.5.4 Crosses between** *D. navojoa* **and** *D. mulleri*

enzyme (Figure 6).

Isozymes are very important in insects and have been used to understand biological problems in several fields of research, including population genetics and systematics, tissue organization, development, metamorphosis, gene regulation and protein synthesis and gene duplication (R. P. Wagner & Selander, 1974). The set of proteins known as esterases constitute one of the most heavily studied groups of isozymes. In the *Drosophila mulleri*  complex, which is the subject of this study, esterases have been extensively studied in several species, including *D. serido* (Lapenta et al., 1995, 1998), *D. buzzatii* (East, 1982; Barker, 1994; Lapenta et al., 1995, 1998), *D. mojavensis* (Zouros et al., 1982; Zouros & Van Delden, 1982; Pen et al., 1984, 1986a, 1986b; Mateus et al., 2009), *D. arizonae* (Zouros et al., 1982; Ceron, 1988; Mateus et al., 2009), *D. aldrichi* (F. M. Johnson et al., 1968; Kambysellis et al., 1968) and *D. mulleri* (F. M. Johnson et al., 1968; Kambysellis et al., 1968; Richardson & Smouse, 1976; Ceron, 1988).

Zouros et al. (1982) detected two esterases with different patterns of temporal and tissuespecific expression in *Drosophila mojavensis* and *D. arizonae* (formerly *D. arizonensis*). They detected a specific β-esterase of the late third instar phase of larval development and in the carcass, named EST-4, in contrast to another β-esterase, named EST-5, which is expressed during all developmental phases and is found predominantly in hemolymph and the fat body. They proposed that the most likely hypothesis is that both enzymes are products of a gene duplication that occurred prior to the speciation of the *D. repleta* group, and their patterns of tissue-specific and temporal expression diverged more recently. This hypothesis was suggested because the enzymes show interlocus heterodimers, different patterns of expression (Zouros et al., 1982) and 82% identity in their N-terminal amino acid sequences

Gene Duplication and Subsequent

study.

study.

Differentiation of Esterases in Cactophilic *Drosophila* Species 365

In our case, Zouros et al. (1982) proposed that the genes coding for EST-4 and EST-5 (*Est-2c* and *Est-2a*, respectively, according to Robin et al., 2009) were also products of a duplication event prior to the divergence of the species that belong to the *D. repleta* group and that the EST-4 gene was later inactivated in some species of this group, including *D. tira*, *D. hydei* and *D. eohydei*. Moreover, the lower activity of EST-4 in *D. mulleri*, *D. aldrichi*, *D. repleta* and *D. peninsularis* could indicate this EST-4 inactivation process. However, our results showed that *D. mulleri*, *D. aldrichi* and *D. wheeleri* had high EST-4 activities compared to the other species (Figure 2A). This difference in the level of activity of EST-4 for the same species in these studies could be the result of differences in the origins of the lines used in each study. Therefore, the populations of *D. mulleri* and *D. aldrichi* that were analyzed by Zouros et al. (1982) could have a certain degree of EST-4 inactivation that was not observed in the present

The enzymes analyzed in this study had biochemical differences compared to other esterases of other *Drosophila* species. For example, the I.P. values for EST-4 and EST-5 for the six *Drosophila* species analyzed were between 6.0 and 7.0 (Table 3). These values were different from those of *D. melanogaster* obtained by Healy et al. (1991), as only 2 out of 15 esterases had I.P. values close to the values obtained in this study (between 6.0 and 7.0). All

Regarding the MWs of these enzymes, our results are in agreement with previous studies that estimated this parameter. EST-4 had MW values between 83 and 89 kD, and EST-5 had MW values between 83 and 94 kD (Table 4), which are very close to the MWs obtained by Pen et al. (1984), which were between 85 and 95 kD for a variant of the EST-4 (with altered specificity to α-naphthyl acetate) using gel filtration chromatography. Pen et al. (1984) also used denaturing gel electrophoresis (SDS-PAGE) and obtained the MWs of the subunits of EST-4 as 62-64 kD. In another study, Pen et al. (1986a) determined the MWs for the subunits of EST-5 as 64-66 kD. Regardless of the method used and the different results obtained (for the entire protein or for subunits), EST-4 had a smaller MW than EST-5, as observed in this

The interspecies crosses performed in this study had results that were in accordance with the known phylogenetic relationships among the species analyzed. This information is based on the morphological work of Throckmorton (1982) and Vilela (1983), the cytological work of Wasserman (1982, 1992 for reviews), several allozyme studies (Zouros, 1973; Richardson et al., 1975; Richardson and Smouse, 1976; Richardson et al., 1977; Heed et al., 1990), molecular studies (Sullivan et al., 1990; Russo et al., 1995; Spicer, 1995, 1996) and an analysis using multiple sources of characters (Durando et al., 2000). The crosses between *D. mulleri* and *D. mojavensis* showed the same results of those of Patterson & Crow (1940) and Bicudo (1982), with offspring obtained only in the direction of *D. mulleri* females and *D. mojavensis* males. For *D. navojoa* crossed with *D. mojavensis,* an F1 was produced only in the direction of *D. navojoa* females and *D. mojavensis* males. In this case, Ruiz et al. (1990) observed descendants in both directions but a very low percentage of offspring, depending on the geographic lineage used, in the direction in which we detected isolation. In crosses between *D. navojoa* and *D. arizonae*, both directions were fertile, which was also found by Ruiz et al. (1990). Finally, in crosses between *D. navojoa* and *D. mulleri,* we detected descendants in both directions, in contrast to the results of Bicudo (1982), who found fertility

only in the direction of *D. mulleri* females and *D. navojoa* males.

others showed values below 6.0, with the majority of values between 4.0 and 5.0.

(Pen et al., 1986a; Pen et al., 1990). More recently, Robin et al. (2009) proposed that, in *D. mojavensis,* these enzymes are most likely encoded by two genes, Est-2a (EST-5) and Est-2c (EST-4), which are products of one gene duplication out of a total of eleven duplications that explain the evolution of the catalytic β-esterase cluster in the *Drosophila* genus (five in the *Sophophora* and 6 in the *Drosophila* subgenus).

Our results reinforce the hypothesis proposed by Zouros et al. (1982), extending the knowledge about these enzymes as products of a gene duplication to other *D. mulleri*  complex species. All six analyzed species show distinct temporal expression patterns for EST-4 and EST-5, with EST-4 showing activity only at the end of the third instar larval stage (Figure 1). The inhibition experiments (Table 2) showed that EST-4 has the same pattern for all six species: it is inhibited by PMSF and not affected by malathion. The opposite was observed for EST-5 in all six species: it was inhibited by malathion and not affected by PMSF. The other inhibitors tested (eserine sulfate, copper sulfate, iodoacetamide and E-64) had no effect on the activity of either enzyme. Moreover, the presence of homozygotes and heterozygotes for EST-5 independent of the EST-4 genotype in *D. navojoa* (Figure 2) and *D. mulleri* (data not shown) support the idea of an independent origin of these enzymes from two distinct loci. Despite these differences, these enzymes display similar features, such as structural similarities (Pen et al., 1986a; Pen et al. 1990) that allow the formation of dimers in *D. mojavensis*, *D. arizonae* and *D. navojoa* (Figure 2).

The gene duplication process is considered one of the most important mechanisms of the generation of new genes and functions during the evolutionary process. Jeffreys & Harris (1982) suggested gene duplication mechanisms that could happen to genes during evolution. Among the mechanisms presented, the most likely mechanism that could have generated the EST-4 and EST-5 loci is the same mechanism that might have generated the globin family, that is, *in tandem* gene duplication by pairing errors during meiosis that cause unequal crossing-over because of the presence of short repeat sequences located in the 3' and 5' ends of the unduplicated ancestral gene.

The EST-5 gene in *D. pseudoobscura* is a good example of gene duplication with later divergence (Brady & Richmond, 1992). The EST-5 enzyme is encoded by the *Est-5B* gene, which is expressed during the life cycles of all insects and is linked to two other genes, *Est-5A* and *Est-5C*, on the X chromosome (Brady et al., 1990). In *D. melanogaster*, the homologous gene is *Est-6*, which codes for the enzyme EST-6 during the insect's life cycle and has only one grouped gene, *Est-P* (Collet et al., 1990). Both *Est-5A* of *D. pseudoobscura* and *Est-P* of *D. melanogaster* are expressed only at the third instar larval stage, producing only one transcript. On the other hand, *Est-5C* of *D. pseudoobscura* is not expressed in any developmental phase (Brady et al., 1990). According to Brady & Richmond (1992), who compared the DNA sequences of coding and flanking regions of all three *D. pseudoobscura* and two *D. melanogaster* genes, only two genes, which are already products of a gene duplication, were present before these two species diverged. These two ancestral genes were probably *Est-5A* and *Est-5B* in the first species and *Est-6* and *Est-P* in the second species. A second duplication occurred later in *D. pseudoobscura*, giving rise to the *Est-5C* gene. However, the findings of Robin et al. (2009) contrast with the evolutionary model proposed by Brady & Richmond (1992); in their analyses, the *Est-5A*/*Est-5B* duplication (which they call *Est6*/*7*) occurred after the melanogaster/obscura group divergence, whereas Brady & Richmond (1992) place this duplication prior to the divergence.

(Pen et al., 1986a; Pen et al., 1990). More recently, Robin et al. (2009) proposed that, in *D. mojavensis,* these enzymes are most likely encoded by two genes, Est-2a (EST-5) and Est-2c (EST-4), which are products of one gene duplication out of a total of eleven duplications that explain the evolution of the catalytic β-esterase cluster in the *Drosophila* genus (five in the

Our results reinforce the hypothesis proposed by Zouros et al. (1982), extending the knowledge about these enzymes as products of a gene duplication to other *D. mulleri*  complex species. All six analyzed species show distinct temporal expression patterns for EST-4 and EST-5, with EST-4 showing activity only at the end of the third instar larval stage (Figure 1). The inhibition experiments (Table 2) showed that EST-4 has the same pattern for all six species: it is inhibited by PMSF and not affected by malathion. The opposite was observed for EST-5 in all six species: it was inhibited by malathion and not affected by PMSF. The other inhibitors tested (eserine sulfate, copper sulfate, iodoacetamide and E-64) had no effect on the activity of either enzyme. Moreover, the presence of homozygotes and heterozygotes for EST-5 independent of the EST-4 genotype in *D. navojoa* (Figure 2) and *D. mulleri* (data not shown) support the idea of an independent origin of these enzymes from two distinct loci. Despite these differences, these enzymes display similar features, such as structural similarities (Pen et al., 1986a; Pen et al. 1990) that allow the formation of dimers in

The gene duplication process is considered one of the most important mechanisms of the generation of new genes and functions during the evolutionary process. Jeffreys & Harris (1982) suggested gene duplication mechanisms that could happen to genes during evolution. Among the mechanisms presented, the most likely mechanism that could have generated the EST-4 and EST-5 loci is the same mechanism that might have generated the globin family, that is, *in tandem* gene duplication by pairing errors during meiosis that cause unequal crossing-over because of the presence of short repeat sequences located in the 3'

The EST-5 gene in *D. pseudoobscura* is a good example of gene duplication with later divergence (Brady & Richmond, 1992). The EST-5 enzyme is encoded by the *Est-5B* gene, which is expressed during the life cycles of all insects and is linked to two other genes, *Est-5A* and *Est-5C*, on the X chromosome (Brady et al., 1990). In *D. melanogaster*, the homologous gene is *Est-6*, which codes for the enzyme EST-6 during the insect's life cycle and has only one grouped gene, *Est-P* (Collet et al., 1990). Both *Est-5A* of *D. pseudoobscura* and *Est-P* of *D. melanogaster* are expressed only at the third instar larval stage, producing only one transcript. On the other hand, *Est-5C* of *D. pseudoobscura* is not expressed in any developmental phase (Brady et al., 1990). According to Brady & Richmond (1992), who compared the DNA sequences of coding and flanking regions of all three *D. pseudoobscura* and two *D. melanogaster* genes, only two genes, which are already products of a gene duplication, were present before these two species diverged. These two ancestral genes were probably *Est-5A* and *Est-5B* in the first species and *Est-6* and *Est-P* in the second species. A second duplication occurred later in *D. pseudoobscura*, giving rise to the *Est-5C* gene. However, the findings of Robin et al. (2009) contrast with the evolutionary model proposed by Brady & Richmond (1992); in their analyses, the *Est-5A*/*Est-5B* duplication (which they call *Est6*/*7*) occurred after the melanogaster/obscura group divergence, whereas Brady &

*Sophophora* and 6 in the *Drosophila* subgenus).

*D. mojavensis*, *D. arizonae* and *D. navojoa* (Figure 2).

and 5' ends of the unduplicated ancestral gene.

Richmond (1992) place this duplication prior to the divergence.

In our case, Zouros et al. (1982) proposed that the genes coding for EST-4 and EST-5 (*Est-2c* and *Est-2a*, respectively, according to Robin et al., 2009) were also products of a duplication event prior to the divergence of the species that belong to the *D. repleta* group and that the EST-4 gene was later inactivated in some species of this group, including *D. tira*, *D. hydei* and *D. eohydei*. Moreover, the lower activity of EST-4 in *D. mulleri*, *D. aldrichi*, *D. repleta* and *D. peninsularis* could indicate this EST-4 inactivation process. However, our results showed that *D. mulleri*, *D. aldrichi* and *D. wheeleri* had high EST-4 activities compared to the other species (Figure 2A). This difference in the level of activity of EST-4 for the same species in these studies could be the result of differences in the origins of the lines used in each study. Therefore, the populations of *D. mulleri* and *D. aldrichi* that were analyzed by Zouros et al. (1982) could have a certain degree of EST-4 inactivation that was not observed in the present study.

The enzymes analyzed in this study had biochemical differences compared to other esterases of other *Drosophila* species. For example, the I.P. values for EST-4 and EST-5 for the six *Drosophila* species analyzed were between 6.0 and 7.0 (Table 3). These values were different from those of *D. melanogaster* obtained by Healy et al. (1991), as only 2 out of 15 esterases had I.P. values close to the values obtained in this study (between 6.0 and 7.0). All others showed values below 6.0, with the majority of values between 4.0 and 5.0.

Regarding the MWs of these enzymes, our results are in agreement with previous studies that estimated this parameter. EST-4 had MW values between 83 and 89 kD, and EST-5 had MW values between 83 and 94 kD (Table 4), which are very close to the MWs obtained by Pen et al. (1984), which were between 85 and 95 kD for a variant of the EST-4 (with altered specificity to α-naphthyl acetate) using gel filtration chromatography. Pen et al. (1984) also used denaturing gel electrophoresis (SDS-PAGE) and obtained the MWs of the subunits of EST-4 as 62-64 kD. In another study, Pen et al. (1986a) determined the MWs for the subunits of EST-5 as 64-66 kD. Regardless of the method used and the different results obtained (for the entire protein or for subunits), EST-4 had a smaller MW than EST-5, as observed in this study.

The interspecies crosses performed in this study had results that were in accordance with the known phylogenetic relationships among the species analyzed. This information is based on the morphological work of Throckmorton (1982) and Vilela (1983), the cytological work of Wasserman (1982, 1992 for reviews), several allozyme studies (Zouros, 1973; Richardson et al., 1975; Richardson and Smouse, 1976; Richardson et al., 1977; Heed et al., 1990), molecular studies (Sullivan et al., 1990; Russo et al., 1995; Spicer, 1995, 1996) and an analysis using multiple sources of characters (Durando et al., 2000). The crosses between *D. mulleri* and *D. mojavensis* showed the same results of those of Patterson & Crow (1940) and Bicudo (1982), with offspring obtained only in the direction of *D. mulleri* females and *D. mojavensis* males. For *D. navojoa* crossed with *D. mojavensis,* an F1 was produced only in the direction of *D. navojoa* females and *D. mojavensis* males. In this case, Ruiz et al. (1990) observed descendants in both directions but a very low percentage of offspring, depending on the geographic lineage used, in the direction in which we detected isolation. In crosses between *D. navojoa* and *D. arizonae*, both directions were fertile, which was also found by Ruiz et al. (1990). Finally, in crosses between *D. navojoa* and *D. mulleri,* we detected descendants in both directions, in contrast to the results of Bicudo (1982), who found fertility only in the direction of *D. mulleri* females and *D. navojoa* males.

Gene Duplication and Subsequent

**5. Conclusions** 

result from a gene duplication event.

**6. Acknowledgments** 

Differentiation of Esterases in Cactophilic *Drosophila* Species 367

To establish the possible role of EST-4, the following information must be considered. Healy et al. (1991) observed that all *D. melanogaster* acetylesterases are inhibited by OTFP (3 octylthio-1,1,1-trifluoropropan-2-one), which is a powerful inhibitor of juvenile hormone esterase activity in Lepidoptera, suggesting that all acetylesterases from this species have similar properties as juvenile hormone esterase. Moreover, East (1982) proposed that esterase-J from *D. buzzatii*, which is supposedly the enzyme from this species that corresponds to EST-4 in this study, is a juvenile hormone esterase, acting together with EST-1 in the larval phase to control the levels of this hormone. In the adult phase, only EST-1 would be responsible for this control. On the other hand, EST-2 could be the enzyme responsible for digestive and detoxification processes and ester absorption in adults. EST-4 has a very tissue-specific and temporal pattern of expression, which indicates that there is a specific regulatory system that controls its expression at a specific tissue (carcass) and period of time (at the end of the larval phase, when all of the processes for pupation have been initiated). Therefore, as an acetylesterase with a very specific temporal expression pattern, EST-4 could be involved in these transformation processes, acting as an auxiliary enzyme for the degradation of juvenile hormone esterase. The degradation of this hormone in this phase allows the liberation of prothoracicotropic hormone by the brain, which stimulates ecdysone production by the prothoracic gland, initiating metamorphosis (Coundron et al., 1981). However, analyzing the EST-4 inhibition data alone could lead to the hypothesis that this enzyme could be a serine-protease that also has esterase activity and is involved in a proteolytic activity during the larva-pupae conversion process; it is likely to be involved in this process. Regarding EST-5, considering the fact that it is expressed during the entire life cycle of the insect and is found mainly in the hemolymph and fat body, it is a

non-specific carboxylesterase that is probably involved in digestive processes.

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

We would like to thank CNPq for funding Rogério P. Mateus (Master's degree fellowship). We would also like to thank CAPES, FINEP and FUNDUNESP for supporting this work,

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 probably belongs to the class of acetylesterases.

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

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

To establish the possible role of EST-4, the following information must be considered. Healy et al. (1991) observed that all *D. melanogaster* acetylesterases are inhibited by OTFP (3 octylthio-1,1,1-trifluoropropan-2-one), which is a powerful inhibitor of juvenile hormone esterase activity in Lepidoptera, suggesting that all acetylesterases from this species have similar properties as juvenile hormone esterase. Moreover, East (1982) proposed that esterase-J from *D. buzzatii*, which is supposedly the enzyme from this species that corresponds to EST-4 in this study, is a juvenile hormone esterase, acting together with EST-1 in the larval phase to control the levels of this hormone. In the adult phase, only EST-1 would be responsible for this control. On the other hand, EST-2 could be the enzyme responsible for digestive and detoxification processes and ester absorption in adults. EST-4 has a very tissue-specific and temporal pattern of expression, which indicates that there is a specific regulatory system that controls its expression at a specific tissue (carcass) and period of time (at the end of the larval phase, when all of the processes for pupation have been initiated). Therefore, as an acetylesterase with a very specific temporal expression pattern, EST-4 could be involved in these transformation processes, acting as an auxiliary enzyme for the degradation of juvenile hormone esterase. The degradation of this hormone in this phase allows the liberation of prothoracicotropic hormone by the brain, which stimulates ecdysone production by the prothoracic gland, initiating metamorphosis (Coundron et al., 1981). However, analyzing the EST-4 inhibition data alone could lead to the hypothesis that this enzyme could be a serine-protease that also has esterase activity and is involved in a proteolytic activity during the larva-pupae conversion process; it is likely to be involved in this process. Regarding EST-5, considering the fact that it is expressed during the entire life cycle of the insect and is found mainly in the hemolymph and fat body, it is a non-specific carboxylesterase that is probably involved in digestive processes.
