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

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

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

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 13; Figure 5).

Gene Duplication and Subsequent

*mulleri*; 2 = *D. navojoa*; 3-9 = hybrid larvae.

both species.

**4. Discussion** 

Smouse, 1976; Ceron, 1988).

Differentiation of Esterases in Cactophilic *Drosophila* Species 363

enzymes in these species (Figure 7). Again, the EST-4 hybrid band migrated closer to the slower band from *D. mulleri*, which could be a consequence of the different I.P. values of these enzymes. The same was not observed for EST-5, as they show similar I.P. values for

Fig. 7. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines of *D. mulleri* and *D. navojoa* and the hybrids obtained from crosses in both directions. 1 = *D.* 

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 &

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

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

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 enzyme (Figure 6).

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

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

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

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 enzymes in these species (Figure 7). Again, the EST-4 hybrid band migrated closer to the slower band from *D. mulleri*, which could be a consequence of the different I.P. values of these enzymes. The same was not observed for EST-5, as they show similar I.P. values for both species.

Fig. 7. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines of *D. mulleri* and *D. navojoa* and the hybrids obtained from crosses in both directions. 1 = *D. mulleri*; 2 = *D. navojoa*; 3-9 = hybrid larvae.
