Esterase-3 polymorphism in the sugarcane borer Diatraea saccharalis (Lepidoptera, Pyralidae)

The migration rate of esterases and their substrate specificity for 4-methylumbelliferyl esters (acetate, propionate, and butyrate) and αand β-naphthyl esters were analyzed in Diatraea saccharalis by starch gel electrophoresis. Substrate preference of esterases was observed with Est-2 and Est-8 isozymes showing substrate specificity for 4-methylumbelliferyl esters and Est-4 isozyme showing specificity for 4-methylumbelliferyl butyrate and α-naphthyl butyrate. Allele variation was detected at the Est-3 locus. Two alleles, Est-3 and Est-3, were identified in pupae with fluorogenic and ester-naphthyl substrates. Chi-square analysis showed no differences between the observed genotypic frequencies and those expected on the basis of Hardy-Weinberg frequencies for the Est-3 locus (χ = 2.4; p < 0.01). The negative value for the Wright’s fixation index (F = -0.2096) calculated for the D. saccharalis population maintained under laboratory conditions indicates an excess of heterozygotes, however, the observed Hardy-Weinberg equilibrium indicates that in the laboratory the population of D. saccharalis behaved as if the moth were randomly mating in nature. The high level of heterozygosity at the Est-3 locus indicates also that this esterase may be a good genetic marker for studies of natural D. saccharalis populations.


Introduction
The sugarcane borer Diatraea saccharalis (Fabricius 1724; Lepidoptera; Pyralidae) is an insect of great economic interest for the sugar-alcohol industry, because it occurs in sugarcane plantations where its larva damages sugarcane by boring into the stalks and reducing sucrose yield (Roc et al., 1981). Due to the peculiar ecological-climatic conditions prevalent in Brazil the sugarcane borer also increases the plants susceptibility to pathogens, allowing invasion by stalk-rot pathogens through larval tunnels, with infection by the red rot fungus (Physalospora tucumanensis) being particularly frequent.
In the warmer tropical climates, where perennial growth of sugarcane is not annually interrupted by spells of cold temperature, chemical control of the sugarcane borer is not recommended and, when practiced, is often ineffective (Roc et al., 1981). In Latin American countries, biological control is the most common method of suppressing D. saccharalis (William et al., 1969;Guagliumi, 1973), and hymenopteran and dipteran insect parasites have long been extensively used for the biological control of D. saccharallis (see review in Conte, 1994). Although no data are available on the genetic structure of D. saccharalis populations, information on genetic variability is especially important for successful large-scale pest control programs and Johnson (1974), has pointed out that enzyme polymorphism may increase the opportunity for adaptability in organisms facing a variable environment.
The present study investigated the electrophoretic profiles of D. saccharalis esterases and obtained preliminary data on the genetic variability present in laboratory colonies of this species which are used for studies of biological control systems based on hymenopteran parasites. Esterase isozymes were chosen because their different forms are genetically determined by several loci, and a high frequency of genetic variants is usually detected in insect populations (Selander, 1976).

Material and Methods
Diatraea saccharalis individuals were obtained from a colony founded by ten adult females and two adult males moths, collected during March and April 1998 in the sugarcane fields of a local producer (The Santa Terezinha Factory, Maringá Region, Iguatemi District, northwest Paraná Genetics and Molecular Biology, 25, 1, 61-64 (2002) Copyright by the Brazilian Society of Genetics. Printed in Brazil www.sbg.org.br State, Brazil), established at the Department of Cell Biology and Genetics, State University of Maringá, Maringá, Paraná, Brazil. The moths had been collected in sugarcane fields infected with this borer and were transferred to the laboratory and maintained in a box (20 x 20 x 10 cm) for further breeding and the establishment of a D. saccharalis colony. To start the laboratory population the larvae, pupae and adult insects were maintained in a nylon-cloth covered wire-cage (50 x 40 x 23 cm) at 24 ± 1°C, 70 ± 10% RH and a 14h photophase (Conte, 1994) and fed with the artificial diet developed by Hensley and Hammond (1968). After eight months, equivalent to about 4 generations of the moths (Conte, 1994), male and female pupae were collected at random and sexually identified under a stereoscopic microscope according to the method of Butt and Cantu (1962).
For electrophoretic analysis individual pupae were homogenized with a glass rod in an Eppendorf tube using 500 µL of a 1% aqueous solution of β-mercaptoethanol and centrifuged at 3,000 rpm for 10 min at room temperature in a model Eppendorf 5417 centrifuge. The supernatants were absorbed with Whatman n. 3 paper strips (5 x 6 mm) which were inserted vertically into a 14% starch gel prepared in 0.02 M Tris-HCl buffer, pH 7.5. In the electrode chambers we used 0.3 M Tris-HCl buffer, pH 7.5. Electrophoresis was carried out at 4°C for about 4-5 h, at 45 mA (9.5 V/cm of gel). The esterase isozymes were stained by the methods of Hopkinson et al. (1973) and Coates et al. (1975) as modified by Ruvolo-Takasusuki et al. (1997), with 4-methylumbelliferyl (4-MUB) esters (acetate, propionate, and butyrate), and αand β-naphthyl acetate and α-naphthyl butyrate as substrates (Tashian, 1969;Ruvolo-Takasusuki et al., 1997). The 4-MUB ester substrate (4 mg) was dissolved separately in 500 µL of acetone and the volume completed to 10 mL using double-distilled water. Following staining with 4-MUB acetate, propionate and butyrate, the gels were incubated for 30-60 min in a solution containing 50 mL of 0.05 M sodium phosphate, pH 6.5, 40 mg Fast Blue RR salt and 4 mL of 1% α-naphthyl acetate, β-naphthyl acetate, and α-naphthyl butyrate, respectively. The αand β-naphthyl esters were dissolved in 50 mL of acetone and the volume completed to 100 mL with double-distilled water. Stained gels were fixed in methanol: double-distilled water: acetic acid (5:5:1) for 3 min and washed with tap water.

Results
The staining techniques revealed the esterase isozyme patterns of the Diatraea saccharalis pupae, and indicated a total of eight esterase loci, referred to in the following text as Est-1 to Est-8. The isozymes Est-2 and Est-8 were substrate specific for 4-MUB esters (acetate, propionate, and butyrate) while Est-4 was specific to 4-methylumbelliferyl butyrate and α-naphthyl butyrate (Figure 1).
Allelic variation was detected at the Est-3 locus, and two alleles, Est-3 F and Est-3 S , were identified in pupae with 4-MUB acetate ( Figure 1A) and αand β-naphthyl acetate (Figure 2). The Est-4 isozyme detected with 4-MUB butyrate ( Figure 1B) and α-naphthyl butyrate was observed migrating between Est-3 F and Est-3 S alleles or sometime showing coincident migration with Est-3 F allele. 62 Ruvolo-Takasusuki et al.  The number of males and females analyzed and the electrophoretic phenotypes of the Est-3 locus are listed in Table 1. Allele frequencies and mean heterozygosities for the Est-3 locus are listed in Table 2. Chi-squared analysis showed no differences between observed genotypic frequencies and the expected Hardy-Weinberg frequencies for the Est-3 locus (χ 2 = 2.4; p < 0.01; Table 3), but the negative value for the Wright's fixation index (Wright, 1965) calculated for the D. saccharalis population maintained under laboratory conditions indicated an excess of heterozygotes.

Discussion
The present study is the first report to relate isozyme polymorphism in the Diatraea saccharalis species. The esterase isozyme patterns indicated a total of eight loci with substrate specificity for 4-MUB esters and α-naphthyl butyrate, and allelic variation was detected at the Est-3 locus.
Despite the method used to establish the D. saccharalis laboratory population which involved enforced inbreeding (Nei, 1975;Hedrich, 1983), the Hardy-Weinberg equilibrium test indicated that random-mating between males and females took place for at least 4 generations in this sub-population. Thus, it is possible to assume that the amount of genetic variation at the esterase loci of D. saccharalis remains constant in the presence of disturbing forces such as bottleneck or founder effects. The study of esterases in laboratory populations of D. saccharalis indicates that the data obtained by Conte (1994) on infestation control by hymenopteran parasites can be applied to natural populations. This is the most important aspect of our study because it shows that the information about genetic diversity in D. saccharalis laboratory populations can support biological control programs aimed at this species, since after being maintained for at least 4 generations under laboratory conditions the moths still behaved as if they were randomly mating in nature.
The Wright's fixation index value indicated an excess of heterozygotes in this D. saccharalis sub-population, reflecting overall outbreeding. The heterozygote excess observed for the Est-3 locus may be attributed to the method used to establish the laboratory population of D. saccharalis, since founder genotypes and random genetic drift are events which can produce higher than expected proportions of heterozygotes, and random drift is a frequent phenomenon in laboratory populations (Falconer and Mackay, 1996).
On the other hand, an overdominance effect can arise at the molecular level since the products of the two alleles can have different properties such as enzymatic activity, heat stability or activity optima for environmental factors such as temperature or pH. The mixture of allozymes may therefore make the heterozygotes more versatile than homozygotes with single allozymes, i.e., they may be less susceptible to the impairment of enzyme function by environmental circumstances (Falconer and Mackay, 1996). Examples of esterase alleles related to specific environmental factors have been reported for other insect groups (Tsakas and Krimbas, 1970;Fournier et al., 1992Fournier et al., , 1993Ono et al., 1994;Dinardo-Miranda and Contel, 1996).
Although few proven cases of overdominance are known (Hartl and Clarck, 1989) and despite the fact that the fitness of the different esterases is unknown, overdominance may be an explanation for the slight excess of heterozygotes in the D. saccharalis sub-population. Heterozygote males and females may have a mating advantage which results in a stable polymorphism phenomenon.
The other important aspect of the present work is that the high level of heterozygosity at the Est-3 locus indicates that this esterase may be a good genetic marker for studies of the D. saccharalis populations.