**1. Introduction**

352 Gene Duplication

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Wilson, T.G., DeMoor, S., Lei, J. 2003. Juvenile hormone involvement in *Drosophila* 

Yamamoto, K., Chadarevian, A., Pellegrini, M. 1988. Juvenile hormone action mediated in male accessory glands of *Drosophila* by calcium and kinase C. Science. 239:916-919. Yang, X.H., Liu, P.C., Zheng, W.W., Zhao, X.F. 2011. The juvenile hormone analogue

Zhang, Z., Xu, J., Sheng, Z., Sui, Y., Palli, S.R. 2011. *Steroid Receptor Co-activator* is required

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Zhu, J., Busche, J.M., Zhang, X. 2010. Identificatino of juvenile hormone target genes in the adult female mosquitoes. Insect Biochemistry and Molecular Biology. 40:23-29. Zhu, J., Chen, L., Sun, G., Raikhel, A.S. 2006. The competence factor beta Ftz-F1 potentiates

tobacco hornworm, *Manduca sexta*. Developmental Biology. 203:233-44. Zhou, X., Riddiford, L. M. 2002. *Broad* specifies pupal development and mediates the 'status

*Methoprene tolerant*. Journal of Biological Chemistry. 286: 8437-8447.

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Phytophagous insects are excellent model systems to study the genetic and ecological bases of adaptation and population differentiation because the host plant constitutes an immediate environmental factor that can affect the early stages of the life cycle (Matzkin, 2005; Matzkin et al., 2006). New host plant exploitation can result in genetic and biochemical adjustments to the new resource and to chemically distinct niches, which can include potentially toxic compounds, new mating environments, parasitoids, bacteria and fungi (Kircher, 1982; Fogleman & Abril, 1990; Via, 1990; Fogleman & Danielson, 2001). These adjustments are the result of a number of physiological changes, including those related to biochemical systems associated with adaptation to the new environment.

The species of the *Drosophila repleta* group occupy different habitats, but their common feature is that they are phytophagous; that is, they lay eggs in rotting cacti cladodes. The developing larvae feed on the yeast that are part of the rotting process (Starmer & Gilbert, 1982; Pereira et al., 1983; Starmer et al., 1986), according to the cactus-*Drosophila*-yeast system; therefore, they are considered specialists. However, adults are generalists because they visit other food sources in their environment (Morais et al., 1994). This ecological specificity of cactophilic *Drosophila* directly influences species distribution, as they are always associated with the host cactus distribution (Tidon-Sklorz & Sene, 1995; Manfrin & Sene, 2006; Mateus and Sene, 2007).

*Drosophila* has been used as a research model for more than a century, and the first report of gene duplication was described by Bridges for the *Bar* locus in *D. melanogaster* over 70 years ago (Bridges, 1936). Since that time, mainly after the advent of biochemical and molecular biology techniques, several other examples of duplicated genes have been presented, and pathways of evolution by gene duplication have been proposed (for example, Stephens, 1951; Nei, 1969). These pathways were thoroughly discussed in 1970 in Ohno's book "Evolution by gene duplication" (Ohno, 1970). Subsequently, several other works have reviewed the mechanisms and roles of gene duplication in the evolutionary process (A. Wagner, 2002; Kondrashov et al., 2002; and Zhang, 2003).

Currently, the genomes of twelve *Drosophila* species have been completely sequenced (Tweedie et al., 2009), but many aspects of the functional divergence of the products of a

Gene Duplication and Subsequent

Table 1.

TX, US).

**2.3 Obtaining hybrids** 

**2.4 Esterase detection** 

Differentiation of Esterases in Cactophilic *Drosophila* Species 355

stocks were multifemales, except for the NA-FS stock, which was isofemale. The laboratory stocks were obtained from Prof. Dr. Hermione E. M. C. Bicudo (Department of Biology, IBILCE/UNESP, São José do Rio Preto, Brazil), who brought them to Brazil from stocks of the Genetics Foundation (University of Texas, Austin, TX, US). The two line stocks were

All laboratory and line stocks were maintained as mass cultures at a constant temperature of 20ºC ±1ºC in culture vials with standard banana medium. The origin of each stock is listed in

*Drosophila mulleri* MU Guayalejo, Tamazunchale, México

*Drosophila arizonae* AR Guayalejo, Tamazunchale, México

Table 1. List of analyzed species, with the respective codes and original localities of the stocks (all stocks were obtained from the Genetics Foundation, University of Texas, Austin,

Late third instar larvae and adult flies were collected directly from the vials and immediately frozen at -20º C for further electrophoretic analyses. The larvae in that phase show yellowish spiraculum and maximum EST-4 activity. Female adult flies were collected

Mass crossings in both directions were performed in population boxes (16 cm3), using 200 couples, between NA-FS and MU-S, NA-FS and MO, NA-FS and AR and MU-S and MO. After setting up a cross, the courtship behavior was observed for 10 minutes, as described by Markow (1981). The culture media were placed in Petri plates at the bottom of the boxes and were changed every three days. After every plate change, the plates were inspected to detect eggs. The plates were maintained at a constant temperature of 20ºC ±1ºC until late third instar hybrid larvae were observed. These larvae were obtained directly from the

Esterase detection was performed using 10% polyacrylamide gel electrophoresis (PAGE), adapted by Ceron (1988) from Davis (1964) and Laemmli (1970). All samples were prepared in 25 µL of 0.1 M Tris-HCl (pH 8.8) buffer containing 10% glycerol, where 10 µL was used in the gels. After electrophoresis, all gels were soaked in 0.1 M phosphate buffer (pH 6.2) for 1 hour at 25ºC. After this period, the gels were stained in solution containing 100 mL of phosphate buffer, 10 ml of n-propanol and 120 mg of Fast Blue RR Salt, where 40 mg of αnaphthyl acetate and 30 mg of β-naphthyl acetate, previously dissolved in 2 ml of acetone, were added. After approximately 1 hour, the staining reactions were stopped in a solution of acetic acid:ethanol:water (1:2.5:6.5 by v:v:v). Because the esterases hydrolyze substrates

*Drosophila wheeleri* WH Arroyo Solloro, Baja California, México

prepared from laboratory stocks through endogamic crosses.

**Species Codes Locality** 

*Drosophila aldrichi* AL Austin, Texas, US

*Drosophila navojoa* NA Navojoa, México

*Drosophila mojavensis* MO Baja California, México

**2.2 Obtaining late third instar larvae and adult flies for electrophoresis** 

at 5-10 days old and were used in electrophoresis for comparative analysis.

plates and frozen at -20ºC for further electrophoretic analyses.

gene duplication event cannot be answered through this method alone. A deeper investigation of genetic differentiation after duplication is possible through molecular and biochemical approaches. These approaches are extremely important because gene duplication followed by functional divergence has been considered the primary mechanism of molecular evolution (Lewis, 1951; Ohno, 1970). Analyses of isozymes have been crucial in this process because they provide, along with cytological studies, evidence for the frequent occurrence of gene duplication during the evolutionary process (Gottlieb, 1982; Hart, 1983).

Esterase is a polymorphic group of isozymes that play important biochemical roles in insects. This group is composed of a heterogeneous set of hydrolytic enzymes that are widely distributed among organisms and that catalyze the hydrolysis of esters, peptides, amides and halides (Walker & Mackness, 1983). They are involved in digestive (Argentine & James, 1995) and reproductive processes (Karotam et al., 1993), the degradation of insecticides (Feyereisen, 1995) and female sex pheromones after male recognition (Vogt & Riddiford, 1981) and in the regulation of juvenile hormone levels (Gu & Zera, 1994). In *Drosophila*, esterases make up a diverse set of enzymes (G. B. Johnson, 1973, 1974), and gene duplication has been used as one explanation for their evolution (Zouros et al., 1982; Pen et al., 1990; Mateus et al., 2009).

Several studies on the changes in esterase activity during development in species of the *D. repleta* group have detected two main β-esterases that show different tissue-specific and temporal expression patterns (Zouros *et al.*, 1982; East, 1982; Pen et al., 1984; Pen et al., 1986a, 1986b; Pen et al., 1990; Mateus et al., 2009). One esterase, named EST-4, is present only in later third instar larvae and early pupae and has a high concentration in the carcass. The other esterase, named EST-5, is present throughout the insect life cycle and occurs predominantly in hemolymph and the fat body (Zouros *et al.*, 1982).

According to Zouros *et al.* (1982), who studied these enzymes in *D. mojavensis* and *D. arizonae*, the most likely hypothesis is that these enzymes are products of a gene duplication as old as the *D. repleta* group that diverged later regarding their patterns of tissue-specific and temporal expression. This hypothesis was suggested because these enzymes show interlocus heterodimers, different patterns of expression (tissue and temporal) and 82% identity in the N-terminal amino acid sequence (Pen *et al.*, 1986a; Pen et al., 1990). More recently, Robin et al. (2009) demonstrated that these enzymes are encoded by two genes that are products of a gene duplication, *Est-2a* (EST-5) and *Est-2c* (EST-4), in *D. mojavensis*.

In this study, we investigated several genetic and biochemical features of EST-4 and EST-5 in six species of the *D. repleta* group, three belonging to the *D. mulleri* cluster (*Drosophila mulleri*, *D. aldrichi* and *D. wheeleri*) and three belonging to the *D. mojavensis* cluster (*D. mojavensis*, *D. arizonae* and *D. navojoa*) of the *D. mulleri* complex, as well as hybrids from crosses involving some of these species. We aimed to establish the biochemical and genetic differences among and possible physiological roles for these enzymes in the metabolic processes of this group of *Drosophila* species.

### **2. Materials and methods**

### **2.1 Species**

The materials used in this study included laboratory stocks of six *Drosophila* species (*D. arizonae* - AR, *D. mojavensis* - MO, *D. navojoa* - NA, *D. mulleri* - MU, *D. aldrichi* - AL and, *D. wheeleri* - WH) and two homozygote line stocks, one for the EST-5 "slow" allele of *D. mulleri* (MU-S) and another for the EST-4 "fast" and EST-5 "slow" alleles of *D. navojoa* (NA-FS). All

gene duplication event cannot be answered through this method alone. A deeper investigation of genetic differentiation after duplication is possible through molecular and biochemical approaches. These approaches are extremely important because gene duplication followed by functional divergence has been considered the primary mechanism of molecular evolution (Lewis, 1951; Ohno, 1970). Analyses of isozymes have been crucial in this process because they provide, along with cytological studies, evidence for the frequent occurrence of gene duplication during the evolutionary process (Gottlieb, 1982; Hart, 1983). Esterase is a polymorphic group of isozymes that play important biochemical roles in insects. This group is composed of a heterogeneous set of hydrolytic enzymes that are widely distributed among organisms and that catalyze the hydrolysis of esters, peptides, amides and halides (Walker & Mackness, 1983). They are involved in digestive (Argentine & James, 1995) and reproductive processes (Karotam et al., 1993), the degradation of insecticides (Feyereisen, 1995) and female sex pheromones after male recognition (Vogt & Riddiford, 1981) and in the regulation of juvenile hormone levels (Gu & Zera, 1994). In *Drosophila*, esterases make up a diverse set of enzymes (G. B. Johnson, 1973, 1974), and gene duplication has been used as one explanation for their evolution (Zouros et al., 1982; Pen et

Several studies on the changes in esterase activity during development in species of the *D. repleta* group have detected two main β-esterases that show different tissue-specific and temporal expression patterns (Zouros *et al.*, 1982; East, 1982; Pen et al., 1984; Pen et al., 1986a, 1986b; Pen et al., 1990; Mateus et al., 2009). One esterase, named EST-4, is present only in later third instar larvae and early pupae and has a high concentration in the carcass. The other esterase, named EST-5, is present throughout the insect life cycle and occurs

According to Zouros *et al.* (1982), who studied these enzymes in *D. mojavensis* and *D. arizonae*, the most likely hypothesis is that these enzymes are products of a gene duplication as old as the *D. repleta* group that diverged later regarding their patterns of tissue-specific and temporal expression. This hypothesis was suggested because these enzymes show interlocus heterodimers, different patterns of expression (tissue and temporal) and 82% identity in the N-terminal amino acid sequence (Pen *et al.*, 1986a; Pen et al., 1990). More recently, Robin et al. (2009) demonstrated that these enzymes are encoded by two genes that

are products of a gene duplication, *Est-2a* (EST-5) and *Est-2c* (EST-4), in *D. mojavensis*.

In this study, we investigated several genetic and biochemical features of EST-4 and EST-5 in six species of the *D. repleta* group, three belonging to the *D. mulleri* cluster (*Drosophila mulleri*, *D. aldrichi* and *D. wheeleri*) and three belonging to the *D. mojavensis* cluster (*D. mojavensis*, *D. arizonae* and *D. navojoa*) of the *D. mulleri* complex, as well as hybrids from crosses involving some of these species. We aimed to establish the biochemical and genetic differences among and possible physiological roles for these enzymes in the metabolic

The materials used in this study included laboratory stocks of six *Drosophila* species (*D. arizonae* - AR, *D. mojavensis* - MO, *D. navojoa* - NA, *D. mulleri* - MU, *D. aldrichi* - AL and, *D. wheeleri* - WH) and two homozygote line stocks, one for the EST-5 "slow" allele of *D. mulleri* (MU-S) and another for the EST-4 "fast" and EST-5 "slow" alleles of *D. navojoa* (NA-FS). All

predominantly in hemolymph and the fat body (Zouros *et al.*, 1982).

processes of this group of *Drosophila* species.

**2. Materials and methods** 

**2.1 Species** 

al., 1990; Mateus et al., 2009).

stocks were multifemales, except for the NA-FS stock, which was isofemale. The laboratory stocks were obtained from Prof. Dr. Hermione E. M. C. Bicudo (Department of Biology, IBILCE/UNESP, São José do Rio Preto, Brazil), who brought them to Brazil from stocks of the Genetics Foundation (University of Texas, Austin, TX, US). The two line stocks were prepared from laboratory stocks through endogamic crosses.

All laboratory and line stocks were maintained as mass cultures at a constant temperature of 20ºC ±1ºC in culture vials with standard banana medium. The origin of each stock is listed in Table 1.


Table 1. List of analyzed species, with the respective codes and original localities of the stocks (all stocks were obtained from the Genetics Foundation, University of Texas, Austin, TX, US).
