Cyclic colonies of S and R strains of malaria vector, Anopheles culicifacies, were reared in an insectary at National Institute of Malaria Research, Delhi maintained at a temperature of 28 ± 1°C and 75 ± 5% relative humidity and fitted with simulated dusk and dawn machine with a photoperiod of 14 hour day and 10 hour night as described by Adak et al. (1999) .
Mosquitoes were fed upon 1% glucose soaked pads and raisins. Female mosquitoes were offered rabbit blood for ovarian development. Following hatching, larvae were reared in enamel trays containing de-chlorinated water and fed on powdered dog biscuits and brewer's yeast tablets in 3:2 ratios.
Cloning of ascp30 of A. culicifacies
Total RNA was isolated from the body tissue of refractory population of A. culicifacies by the TRIZOL Reagent method according to the manufacturer's instructions (Invitrogen, USA). To avoid contamination of the digestive proteases, the gut was removed from the mosquitoes aseptically. Up to 5 μg total RNA was used for cDNA synthesis using reverse transcriptase enzyme (Superscript II) and adapter primer (AP, 5' GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT 3').
Degenerate primers, SerPr1 (5'-TGG GTC/G C/GTG ACC/G GCC/G GCG/A/T/C CAC/T-3') and SerPr2 (5'-ACG AGC/G CGA/G CCA/G CCC/G GAA/G TCG/A/T/C-3') were designed based on the sequence of reported serine protease in A. gambiae . Using these degenerate primers and cDNA as template, a 450 bp fragment was amplified. The fragment was cloned into pGEM-Teasy vector (Promega Corporation, USA) and sequenced by Microsynth (Switzerland). On the basis of sequence of cloned insert, a set of gene-specific primers, DSerPr1 (5'-CGT GCA CTT GAA CAT TAT CTC-3') and DSerPr2 (5'CTG GTT GGC AGC GTC ACG A-3') were synthesized, to obtain 5' and 3' ends of the gene respectively, by RACE (rapid amplification of cDNA ends) according to the protocol described in the 5' and 3' RACE kit manuals (Invitrogen). Gene-specific end primers, 10FMOS (5'-ATG AAA CTG TTC ATC GTC GT-3') and 11R (5'-TTC AGT ACT TGA TGC CAG ATT-3') were designed and synthesized on the basis of sequences of 5' and 3' RACE fragments to obtain the full-length serine protease gene. Using these end primers and cDNA as template, a complete 816 bp gene (ascp30) was isolated, cloned in pGEMT-easy vector and sequenced. All the primers were synthesized from Microsynth.
Dissections and RNA extractions
The mosquitoes were dissected on clean new slide in a drop of ice-cold sterile DEPC-treated water and body tissue (abdomen and thorax) was collected by removing midgut, heads, legs and wings. Body tissue from ten mosquitoes was pooled in 500 μl of TRIZOL. Mosquito larvae of the different instars were also collected and stored in TRIZOL at -70°C.
The tissues were ground to homogeneity with a battery-driven hand-held homogenizer using DEPC-treated sterile grinder tips and processed for RNA extraction by TRIZOL Reagent method. Contaminating DNA was removed by treatment with RQ1 DNase (Promega Corporation) according to manufacturer's instructions. The total RNA isolated was used as a template for semi-quantitative and real time RT-PCR analysis of acsp30 transcripts using β-actin as internal control.
Blood feeding strategy
Rodent malaria parasite, Plasmodium vinckei petteri 279BY, supplied by Prof. Irene Landau, Museum National d'Historie Naturelle, Paris , was maintained on rodent-mosquito cycle at the National Institute of Malaria Research, Delhi . Cryo-preserved Plasmodium vinckei petteri infected blood isolates (500 μl) were mixed with an equal volume of incomplete RPMI culture media and were inoculated into healthy 4–5 weeks old Balb/c mice. Thin blood smears prepared from peripheral blood of infected mice were fixed in methanol and stained in JSB stain . The slides were examined under oil immersion lens of compound microscope (Carl-Zeiss, Germany) for the presence of various stages of parasite. Ideal time for feeding was ascertained by periodically monitoring the parasitemia of infected mice and looking for the presence of at least 0.5% mature gametocytes. Four- to six-day old mosquitoes were starved by depriving them of raisin and glucose pads for nine hours. Approximately 100–200 starved mosquitoes were held in cages and gametocyte-positive mouse was placed in the cage for 1 hour during daytime for feeding. Subsequently, the full-fed mosquitoes were separated from unfed and partially-fed mosquitoes in a separate cloth cage labeled accordingly and were maintained at 23–24°C temperature and 65–70% relative humidity in the insectary. Mosquitoes were dissected after 6, 12, 18 and 24 hours of blood feeding and the tissues were stored in TRIZOL at -70°C until analysis.
Semi-quantitative RT-PCR analysis
The relative transcript abundance of acsp30 in naïve mosquitoes (S and R strains) was determined by semi-quantitative RT-PCR using a One-step RT-PCR kit (Qiagen GmbH, Germany). The amount of RNA in both the tissues was normalized to the β-actin gene. The sequences of the primer pairs used for amplifying β-actin were β-actinFor (5'-CAG ATC ATG TTT GAG ACC TTC AAC-3') and β-actinRev (5'-GA/C/TC CAT CTC C/TTG CTC GAA A/GTC-3'). Using the normalized amount of RNA as template, ascp30 transcript was amplified by using gene-specific primers, forward 10F (5'-ATCAGTTACCAATCGATCTTGCC-3') and reverse 4R (5'-CGTACGTTCCCATGCACTG-3') to obtain a 500 bp amplicon. The amplification regimen was as follows: reverse transcription at 48°C for 30 minutes, inactivation at 95°C for 15 minutes and the PCR at 94°C for 30 seconds, 52°C for 30 seconds and 72°C for 30 seconds for 33 cycles followed by a final extension of 72°C for 10 minutes. Before loading on 1% agarose gel, the RT-PCR products were treated with 1 μg of RNase (Qiagen GmbH) at 37°C for 10 minutes to eliminate template RNA. The gel was photographed with Polaroid 667 black and white print film.
Real Time RT-PCR analysis
Real time RT-PCR was performed using Quanti Tect SYBR Green RT-PCR kit (Qiagen GmbH) and iCycler TM system (Bio-Rad Laboratories, USA) to measure relative transcript levels of acsp30 in naïve R and S mosquitoes, in various developmental stages of R strain mosquito and after blood-feeding and parasite-feeding treatments. The Primer 3 web-based tool was used to design gene-specific primers, ensuring that the length of the PCR product was 300 bp. β-actin amplicon was obtained by using β-actinFor and β-actinRev primers and acsp30 amplicon was obtained by using AcSp30For (5'-GTC AGA CCG CTG GTG GTA AT-3') and AcSpRev (5'-CTC ACG GTT GAG GAA CGT CT-3') primers. Each 25 μl reaction mixture contained 2 μl of template RNA (100–500 ng/μl), 2× QuantiTect SYBR Green RT-PCR Master Mix, 2 μl of primers (5 pmoles/μl), RNase-free water and 0.25 μl of QuantiTect RT enzyme Mix. Real-time cycler conditions included a preliminary reverse transcription at 48°C for 30 minutes, an initial activation step at 95°C for 15 minutes and 40 cycles of denaturation (94°C), annealing (52°C) and extension (72°C) for 30 seconds each. The final step included gradual temperature increase from 50°C to 94°C at the rate of 1°C/10 seconds to enable melt-curve data collection. A non-template control (NTC) was run with every assay. The threshold cycles (CT) were recorded for acsp30 and β-actin amplicons during each experiment. Difference between the CT of β-actin and acsp30 or ΔCT was determined and the relative abundance of acsp30 was calculated in different treatments using Comparative CT method using the formula 2-ΔΔCT.
Isolation of genomic clone of acsp30
Genomic DNA (gDNA) was isolated from the body tissue of R and S strains of mosquito by the method of Henry et al., 1990 . Using gDNA as template and end primers, 10FMOS and 11R, the genomic clones of acsp30 from both strains were amplified by PCR. The obtained amplicons were cloned into pGEM-Teasy vector, sequenced by ABI PRISM and a comparative analysis of the nucleotide sequence from both strains was done using the ClustalW (MacVector Version 7.0).
Isolation and cloning of upstream regulatory sequences
Three nested gene-specific reverse primers; R1 (Biotinylated) (5'-ACTACGAGGAGATTCACGCATGG-3'), R2 (5'-TTA TTCACACCTTGAGTTCAATTG-3') and R3 (5'-GGCTAAAACGACGACGATGAACAG-3') were designed from the 5' end of serine protease cDNA sequence. Using 50 ng of genomic DNA as template, PCR was performed in four different tubes with four forward walker primers (WP) [WP1, 5'-CTAATACGACTCACTATAGGGNNNNATGC-3'; WP2, 5'-CTAATACGACTCACTATAGGGNNNNGATC-3'; WP3, 5'-CTAATACGACTCACTATAGGGNNNNTAGC-3'; WP4, 5'-CTAATACGACTCACTATAGGGNNNNCTAG-3'] and the reverse biotinylated primer, R1 according to the directional walking method described by Mishra et al., 2002 . Amplified biotinylated products were then immobilized on streptavidin-linked paramagnetic beads and non-biotinylated DNA was washed off. Nested PCR was carried out using the above immobilized PCR product as template and gene-specific primer, R2 and T7-based forward walker primer (5'-CTAATACGACTCACTATAGGG-3'). Amplified PCR products were cloned into pGEM-Te vector and sequenced by Microsynth.
Forward primers containing the Kpn I site were designed and synthesized. They were located at 702 bp and 333 bp upstream to the translational start site (ATG) at +1 position. These included; RefDelFor702, (5'-GGTACCCAATGACGCGTTAAGCC-3'), RefDelFor333, (5'-GGTACCGATAAGAACGACCTGG-3') for the refractory strain and SusDelFor702, (5'-GGTACCTCCTGATCAACTATAT-3'), SusDelFor333, (5'-GGTACCGATAAGAACGAC CTG G-3') for the susceptible strain. The reverse primers were designed from a region just upstream to the ATG and contained Xho I restriction site. These included, RefSPRev, (5'-CTCGAGGATTAG TACTGACCACTACGG-3') for the R strain and SusSPRev, (5'-CTCGAGGATTAGTACTGACCTCTACGG-3') for the S strain. Using the DNA of the pGEM-Teasy clones as template and the above primers, a 32 cycle PCR was carried out with the following conditions; 94°C for 30 seconds, 52°C for 30 seconds and 72°C for 1 minute with a final extension at 72°C for 5 minutes. PCR products were again cloned into pGEM-Teasy vector. Plasmids prepared from the positive colonies were double digested with Kpn I and Xho I. The fallout was eluted from the gel using Qiagen gel extraction kit and ligated into promoter-less luciferase reporter vector pGL3 Basic (Promega Corporation), which was pre-digested with Kpn I and Xho I enzymes. Resulting reporter plasmids were named pGL3-Ref702, pGL3-Sus702, pGL3-Ref333 and pGL3-Sus333.
Computer-based sequence analysis
The upstream sequences of acsp30 isolated from R and S strains were analyzed for potential transcription factor binding sites using the MatInspector program ; matrix library was set as Insect library, the core similarity at 0.75 and optimized matrix similarity was used for both the strains. The upstream sequences for the two strains were aligned using the ClustalW program (MacVector Version 7.0) and EMBOSS alignment programs. The core promoter elements and other vertebrate transcription binding factors were identified using reported consensus sequences as described in the results.
Cell culture, transfections and luciferase assay
Drosophila S2 cells were obtained from Invitrogen and were maintained at 27°C in Schneider's Drosophila medium (Invitrogen) with 10% heat inactivated fetal bovine serum in 25 cm2 cell culture flasks. One day before transfection, S2 cells were seeded at 0.75 × 106 cells/ml in 24-well tissue culture plates to achieve 80% confluency. DNA (1.5 μg) was transfected using cellfectin (Invitrogen) according to the manufacturer's instructions. The transfection was carried out in serum-free medium for 8–12 hours. The transfection medium was then replaced with serum-containing medium and the cells were harvested 48 hours after transfection in 50 μl of 1× Cell Culture Lysis Reagent. Luciferase activity was then measured using the Luciferase assay system (Promega Corporation) and the luminescence was read on a Packard LumiCount manual luminometer. A pGL3-Control vector containing luciferase reporter under control of SV40 promoter served as a control. Each transfection was repeated three times and the luciferase activity was measured twice for each sample.
Preparation of nuclear extracts
Five-day old, adult female mosquitoes were dissected on a clean slide in a drop of ice-cold sterile phosphate-buffered saline (PBS) pH 7.4 under a dissecting microscope. The midgut, head, legs and wings were removed and the body tissue of 100 to 200 mosquitoes from both R and S strains were pooled and stored under liquid nitrogen until required. The tissues were ground to homogeneity in a minimum volume of PBS, with a battery-driven hand-held homogenizer using sterile grinder tips and processed for nuclear protein extraction according to the protocol of Lin et al., (2004)  with minor modifications. Briefly, homogenized tissue was solubilized in buffer A (10 mM HEPES, 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM Na3VO4, 10% glycerol and 1 μg/ml of aprotinin, pepstatin, and leupeptin (pH 7.9) for 30 min at 4°C by gentle rocking on a nutator and centrifuged at 12,000 × g at 4°C for 1 min. The supernatant containing the cytoplasmic proteins was discarded and the pellet was then extracted with buffer B (20 mM HEPES, 350 mM NaCl, 10 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM Na3VO4, 0.2% Nonidet P-40, 10% glycerol, 1 μg/ml aprotinin, pepstatin and leupeptin, pH 7.9) at 4°C for one hour by gentle rocking on a nutator as described above. The extracts were centrifuged at 13,000 × g at 4°C for 5 minutes and the supernatants (nuclear extract) were snap frozen in liquid N2 and stored at -70°C for subsequent use in EMSAs. All the protein concentrations were estimated by the method of Bradford (1976) .
Electrophoretic mobility shift assay (EMSA)
Primers were designed to encompass the 400 bp upstream region (-702 to -302 bp) of acsp30 that was responsible for difference in promoter strength in the two strains. Three different fragments (100 bp, 188 bp and 400 bp) from upstream regions of R and S strains were selected for EMSAs. The DNA probes for EMSAs were prepared by amplifying and radioactively labeling the fragments by PCR in a 50 μl reaction mixture. The reaction consisted of 10 ng template (upstream regions from R and S), 10 pmol of each primer, 100 μM dNTPs, 10 μl α-32P-dCTP (10 mCi/ml, 3000 Ci/mmol, PerkinElmer, USA), and 2.5 units of Taq DNA polymerase (Clontech, USA). The PCR included 32 cycles of 30 s denaturation at 94°C, 30 s annealing at 52°C and 30 s extension at 72°C, followed by a final extension of 5 min at 72°C. Labeled probes were purified using a QIAquick nucleotide removal kit (Qiagen GmbH) and the counts were checked by scintillation counter (Beckman, USA).
The probes were named based on the size of the amplified DNA fragment. In the R strain, R100 probe (-702 and -602 bp) was amplified using RefUp702For (5'-CAA TGA CGC GTT AAG CCT GAT-3') and RefUp602Rev (5'-GAT CGC CGT CGT CCA TCA ACA-3') primers; R188 probe (-702 and -514 bp) by RefUp702For and RefUp514Rev (5'-TTC ATG TGG TTT CAT GAT TTA TTA-3') primers; R400 probe (-702 and -302 bp) by RefUp702For and RefUp302Rev (5'-CTT TCG AAT GAC GCC AGG T-3') primers. Similarly for the S strain, S100 probe (-702 and -602 bp) was amplified using SusUp702For (5'-TCC TGA TCA ACT ATA TGG GTT CCT-3') and SusUp602Rev (5'-CTA TTG AAA GAA TCA ATT TGC TAA-3') primers; S188 probe (-702 and -514 bp) by SusUp702For and SusUp514Rev (5'-TTC ATA TCT CAT CAT TTA TTA AAA ATT-3') primers; S400 probe (-702 and -302 bp) by SusUp702For and SusUp302Rev (5'-ATT TCG TAA TGA CGG CCA GGT-3') primers. EMSAs were performed using 10 to 20 μg of nuclear extract from either R or S and incubated at 37°C for 25 minutes with 32P-labeled probes (30,000 c.p.m. per reaction) and 2 μg of poly (dI-dC) (Sigma). The binding buffer contained 20 mM HEPES (pH 7.9), 1 mM dithiothreitol, 1 mM EDTA (pH 8.0), 1.5 mM MgCl2 and 4% glycerol, in a final volume of 20 μl. For competition experiments, a 100-fold molar excess of unlabeled probe was pre-incubated in the reaction mixture at 37°C for 10 min before labeled probe was added. The DNA-protein complex was resolved on a 2% agarose gel (pre-run for 1 h at 4°C) in 0.5× Tris-borate EDTA (TBE) buffer at 200 V at 4°C. Gels were dried and exposed for autoradiography. Scanning was performed using the Typhoon 9210, Variable Mode Imager from Amersham Biosciences, USA. The intensities of the signals were quantified by the Image Analysis Software, ImageQuant TL (Amersham Biosciences) and represented as the percentage ratio of S to R signal intensities. The accession number of the serine protease gene is [GeneBank:AY995188].