The glutathione transferases (GSTs) are a large family of multifunctional enzymes involved in the detoxification of a wide range of xenobiotics including insecticides (Enayati et al., 2005). GSTs can metabolize insecticides by facilitating their reductive dehydrochlorination or by conjugating glutathione (GSH; L-glutamyl-cysteinyl-glycine) to xenobiotic compounds with electrophilic centers (e.g. drugs, herbicides, insecticides), converting them from reactive lipophilic molecules into water-soluble non-reactive conjugates that may easily be excreted (Chen et al., 2003). In addition, they contribute to the removal of toxic oxygen free radical species produced through the action of pesticides.
There are at least two ubiquitously distributed distantly related groups of GSTs, classified according to their location with in the cell: microsomal and cytosolic. A third group of GSTs, the Kappa class, are located in mammalian mitochondria and peroxisomes (Lander et al., 2004; Morel et al., 2004) and are structurally distinct from the microsomal and cytosolic GSTs (Robinson et al., 2004). A single microsomal GST gene is present in the genome of the fruit fly Drosophila melanogaster whereas the mosquito Anopheles gambiae has three microsomal GST genes (Ranson et al., 2002; Toba and Aigaki, 2000). Microsomal GSTs have not been implicated in the metabolism of insecticides (Enayati et al., 2005). Insect cytosolic GSTs were initially assigned numbers according to their order of elution from the various purification procedures employed or isoelectric points (Prapanthadara et al., 1993; Clark et al., 1985).
Over 40 GST genes have been detected in the genomes of higher eukaryotes for which full genome sequence data are currently available (Holt et al., 2002). These have been classified into at least 13 different classes based on their amino acid sequence identities, immunological properties and, where known, substrate specificities (Ortelli et al., 2003). The majority of studies on insect GSTs have focused on their role in conferring insecticide resistance (Ding et al., 2003; Vontas et al., 2001) and, more recently, in protecting against cellular damage by oxidative stress (Zou et al., 2000; Singh et al., 2001). Elevated GST activity has been associated with resistance to all the major classes of insecticides (Vontas et al., 2001; Huang et al., 1998; Prapanthadara et al., 1993). Insect GSTs were recently classified into six classes (d, e, s, q, w and z) by comparative analysis of the D. melanogaster and An. gambiae genomes (Chen et al., 2003). The two largest GST classes in An. gambiae are the insect specific delta (d) and epsilon (e) classes (Ding et al, 2003).
In this study we analyzed and compared the nucleic acid and amino acid sequences of the GSTe2 gene in specimens of Anopheles stephensi which were collected from the Saravan, Chabahar, Nikshahr districts in the Sistan and Baluchistan province (exposed to insecticide spraying), and the Kazeroon district in the Fars province (representing a control area) where insecticides have not been applied for the last 30 years. GSTe2 encodes an enzyme that has the highest levels of Dichloro-Diphenyl-Trichloroethane (DDT) dehydrochlorinase activity (Enayati et al., 2005; Ortelli et al., 2003).
MATERIALS AND METHODS
Mosquito collection, morphological identification and susceptibility test: Specimens of An. stephensi, collected from Saravan, Chabahar, and Nikshahr districts in the Sistan and Baluchistan province (under insecticide treatment) and Kazeroon district in the Fars province (control area), were identified by using the morphological key to Iranian anophelines (Shahgoudian, 1960). Susceptibility tests were carried out in two replicates with standard WHO impregnated paperas of DDT 4%, dieldrin 0.4 %, malathion 5%, permethrin 0.25 %, lambda cyhalothrin (ICON) 0.1 %, deltamethrin 0.025 % on An. stephensi specimens, based on recommended procedures. Mortality counts (percent of death) were recorded after 24 h recovery period.
DNA extraction and PCR amplification of the GSTe2 gene: DNA was extracted from collected specimens by using a slight modification of the Collins’ method (Collins et al., 1987). Application of PCR for the amplification of GSTe2 including exon I and II and full sequence of intron I was carried out on all specimens by E2f (forward) (5´-ATCACCGAGAGCCACGCAATCAT-3´) and E2r (reverse) (5´-GCCACCGTTCGCTTC CTCGTAGT-3´) primers which were designed on the basis of the An. gambiae genome. PCR conditions were optimized by changing the thermal cycling conditions (ramp time and annealing temperature). The optimal conditions for amplification of the GSTe2 gene were as follows: 35 cycles of denaturation at 95ºC for 1 min , annealing at 62ºC for 1 min and extension at 72ºC for 1min with a 10 min extra extension in the last cycle. Specimens of An. stephensi from the Fars province and An. gambiae (LSTM strain) were used as positive and control DNA samples, respectively, while the negative control was a 24 ml PCR mixture plus 1 ml of ddH2O instead of DNA. 5 ml of each of the amplification products was mixed with loading buffer and subjected to electrophoresis in 1.5% (w/v) agarose gel in TBE buffer containing ethidium bromide. The resulting fragments were visualized by a UV transilluminator (Uvitec, Uk). Selected PCR products were purified from the gel by using the QIAquick gel extraction kit (Qiagen, Germany).
Sequence analysis: Sequencing of specimens was performed on an AB1 sequencing machine (Applied BioSystem, USA) from both sides of the amplified fragments using forward and reverse primers. Sequence data, on arrival were double checked by comparison with the related signals and blast analysis. Gene Runner (Version 3.05, Hastings Software Inc., Hastings on Hudson, NY) and ClustalW (Thompson et al., 1994) programs were used for determination of amino acid sequences, detection of reading frames, and alignments.
The WHO routine susceptibility test carried out in duplicate during this study showed that An. stephensi specimens collected in areas under insecticide treatment for vector control (Saravan, Chabahar and Nikshahr districts in the Sistan and Baluchistan province) were resistant to DDT (39% death in the presence of 4 % (g/m2) DDT), while specimens from the control area with no insecticide application since the last 30 years (Kazeroon district in the Fars province) showed no resistance to DDT. Furthermore, resistance to the other tested insecticides dieldrin 0.4%, malathion 5%, permethrin 0.25%, Icon 0.1%, Deltamethrin 0.025% (g/m2) were not detected in any of the examined An. stephensi specimens.
Amplification of the GSTe2 region by E2f and E2r primers in An. stephensi specimens from the Sistan and Baluchistan and Fars provinces revealed 492 and 489 bp fragments on 1.5% (w/v) agarose gel (Fig. 1). These fragments were purified and sequenced from both ends. Sequence alignment of the GSTe2 gene including exon I, exon II and intron I, in these An. stephensi specimens showed 98% and 97% similarities between the Kazeroon and Nikshahr populations and/or other populations of the Sistan and Baluchistan province (Saravan and Chabahar), respectively (Fig. 2). The main difference between these sequences is related to intron I region, so that the similarity percentage with respect to the coding sequence of the GSTe2 gene increases to 99% without considering the intron sequence, between the Kazeroon population and three Sistan and Baluchistan populations (Saravan, Chabahar and Nikshahr).
Comparison of the nucleotide sequence of GSTe2 from An. stephensi populations with the main world malaria vector, An. gambiae revealed 86% homology, while amino acid similarity between the two species was approximately 90%. Analysis of the intron I region of the GSTe2 gene in the three populations of An. stephensi showed that the size of this region was 78 and 75 bp in specimens from the Kazeroon district and the three districts of Sistan and Baluchistan province, respectively. Comparisons of the sequence similarity percentages at intron I of the GSTe2 gene among An. stephensi specimens of the Kazeroon, Saravan, Chabahar and Nikshahr districts are shown in Table 1. Nucleotide variation was observed within the Kazeroon and Nikshahr populations in three positions of exon 1 (21, 105, and198). However in intron region, this variation was detected in three corresponding positions (8, 10, and 33) of Kazeroon specimens in compare to Nikshahr population. Moreover, whilst comparing the Kazeroon population with other populations of the Sistan and Baluchistan (Saravan and Chabahar) province, nucleotide differences at one and three positions in exon I (105) and exon II (21, 90, 198) and four positions in intron I (8, 16, 33, 72) of Kazeroon specimens were revealed. Analysis of amino acid sequences in different specimens showed that the nucleotide substitutions in the various strains did not lead to any change in the composition of amino acid sequences. Thus, the resistance status observed after the application of the WHO susceptibility test could be related to over expression of the GST gene. Comparisons of nucleotide variations and amino acid replacements between specimens of the control area and areas under insecticide treatment are shown in Table 2. All of the nucleotide variations occur in the third position and therefore based on the wobble hypothesis, do not cause a change in the amino acid codon.
Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. The observation that the genomic location of a cluster of GST genes coincides with a region of the genome containing a major locus conferring DDT resistance was the main aim of this study which analyzed and compared GSTe2 sequences between susceptible and resistant populations of An. stephensi, one of the most important malaria vectors present in the Middle East and Indian subcontinent. This has now led to identification of mutations in both exons and intron I of the GSTe2 gene in various strains. However, it has been observed that none of these nucleotide substitutions results in any amino acid change, eventhough the WHO susceptibility tests confirm DDT resistance in An. stephensi specimens collected from the Sistan and Baluchistan province. Furthermore, in a comparative study, Dinparast et al. (2006) have shown that the GSTe2 coding sequences of An. stephensi, An. culicifacies, and An. fluviatilis show 82 to 86% similarity at the nucleic acid levels with that of An. gambiae. In their study, species-specific differences have also been detected in intron I of the GSTe2 gene, probably useful as a molecular marker for species-specific identification.
The data from this investigation will be used in future studies to express the protein of this gene in different populations consisting of the three major malaria vector species, An. stephensi, An. culicifacies, An. fluviatilis, in Iran and neighboring countries. This may help in understabding the mechanism underlying the resistance of these important but neglected malaria vectors to DDT and pyrethroids, as have already been shown in An. gambiae and Aedes aegypti (Lumjuan et al., 2005). In addition, eventual characterization of recombinant GSTe2 may shed light on other features of GST, such as glutathione peroxidase activity, which has been detected in Ae. aegypti, but not in An. gambiae.
In conclusion, it can be assumed that the out coming results of this study may serve as first report and baseline data regarding the structure of the GSTe2 gene, including exon I, exon II and intron I, in susceptible and resistance field specimens of An. stephensi. Meanwhile the integration of these data into the malaria control programs will still remain a challenge in Iran and neighboring countries, especially Afghanistan and Pakistan.
This work received financial support from WHO/EMRO (grants ID SGS108/1 and SGS108/3); Pasteur Institute of Iran (grant ID 180; Deputy for Research, Ministry of Health; Center for Diseases Management and Control (CDMC). We also appreciate the kind cooperation of the Zahedan University of Medical Sciences (ZUMS), especially Dr. M. T. H. Tabatabaei, and the Department of Public Health in Zahedan, Chabahar, Saravan, Khash, Nikshahr districts and the Kazeroon Public Health Center.