J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 71 Original Article Resistance Mechanisms of Anopheles stephensi (Diptera: Culicidae) to Temephos Aboozar Soltani 1, 3, *Hassan Vatandoost 3, *Mohammad Ali Oshaghi 3, Naseh Maleki Ravasan 3, Ahmad Ali Enayati 2, Fatemeh Asgarian 2 1Department of Medical Entomology and vector control, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran 2School of Public Health and Health Research Centre, Mazandaran University of Medical Sciences, Sari, Iran 3Department of Medical Entomology and Vector Control, School of Public Health and National Institute of Health Research, Tehran University of Medical Sciences, Tehran, Iran (Received 9 Apr 2014; accepted 24 May 2014) Abstract Background: Anopheles stephensi is a sub-tropical species and has been considered as one of the most important vector of human malaria throughout the Middle East and South Asian region including the malarious areas of south- ern Iran. Current reports confirmed An. stephensi resistance to temephos in Oman and India. However, there is no comprehensive research on mechanisms of temephos resistance in An. stephensi in the literature. This study was de- signed in order to clarify the enzymatic and molecular mechanisms of temephos resistance in this species. Methods: Profile activities of α- and ß-esterases, mixed function oxidase (MFO), glutathione-S-transferase (GST), insensitive acetylcholinesterase, and para-nitrophenyl acetate (PNPA)-esterase enzymes were tested for An. stephensi strain with resistance ratio of 15.82 to temephos in comparison with susceptible strain. Results: Results showed that the mean activity of α-EST, GST and AChE enzymes were classified as altered indi- cating metabolic mechanisms have considerable role in resistance of An. stephensi to temephos. Molecular study using PCR-RFLP method to trace the G119S mutation in ACE-1 gene showed lack of the mutation responsible for organophosphate insecticide resistance in the temephos-selected strain of An. stephensi. Conclusion: This study showed that the altered enzymes but not targets site insensitivity of ACE-1 are responsible for temephos resistance in An. stephensi in south of Iran. Keywords: Anopheles stephensi, temephos, mechanisms of resistance, Acetylcholinesterase gene, malaria Introduction Malaria still remains as a public health prob- lem in the world. Southern parts of Iran are involved with this problem (Vatandoost et al. 2010). Anopheles stephensi is a sub-tropical species and also an important vector of human ma- laria throughout the Middle East and South Asian region, including the Indo-Pakistan sub- continent, with a westward extension through Iran and Iraq into the Middle East and Ara- bian Peninsula. This species is considered to be the main malaria vector in the Persian Gulf area (Oshaghi et al. 2006a and 2006b). Pre- vious studies have shown An. stephensi to be the most prevalent anopheline species in the malarious areas of southern Iran (Vatandoost et al. 2004, Hanafi-Bojd et al. 2012). Temephos, a most widely used organophos- phorus insecticide, has been included in the list of World Health Organization (WHO) as a suitable and safe mosquito larvicide that can be used even in drinking water for con- *Corresponding author: Prof Hassan Vatandoost, E- mail: hvatandoost1@yahoo.com, Dr Mohammad Ali Oshaghi, E-mail: oshaghima@yahoo.com http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 72 trolling of the most mosquito vectors .The toxicity of this insecticide is low and unlike- ly to present acute hazard for human (WHO 2006). Temephos (EC 50%) has been used for some years for larval control program of malaria in Southern Iran (Vatandoost et al. 2006). Many studies on the susceptibility level of An. stephensi to various pesticides have been done in Iran and other countries. Resistance of An. stephensi to different insec- ticide was reported from around the world (Vatandoost et al. 1996). Different levels of resistance to larvicides were reported in anopheline malaria vectors worldwide. Anoph- eles stephensi has an extensive resistance com- paring to other species and is resistant or tol- erant to fenitrothion, temephos and fenthion in India, fenitrothion and pirimiphos-methyl in Iraq, fenitrothion, pirimiphos-methyl, chlorfoxim and foxim in Iran and fenitrothion in Pakistan (Vatandoost and Hanafi-Bojd 2005a). Resistance of other anopheline mos- quito such as An. dthali to temephos also was reported (Hanafi-Bojd et al. 2006). In 2006 for the first time in the Middle East, resistance to temephos was confirmed in An. stephensi breeding in water storage tanks in the Al-Dhahira region of Oman (Anderasen 2006). The level of resistance was 2.5 times higher than that of the WHO diagnostic dose (0. 25 mg/l). However, there was no confirmed report of resistance of An. stephensi to temephos in Iran. Previous stud- ies in Iran showed that this species was com- pletely susceptible to temephos at the WHO diagnostic dose (Vatandoost et al. 2004, Vatandoost and Hanafi-Bojd 2005a, Vatandoost et al. 2005b, Vatandoost et al. 2006). One of the most important molecular mech- anisms of resistance to organophosphate in- secticide in mosquitoes is structural muta- tions that occur in acetylcholinesterase gene. In mosquitoes two cholinesterase genes are existed (ACE-2 and ACE-1). ACE genes have been cloned from the mosquitoes Aedes aegypti and An. stephensi, both of these genes are also sex linked (Hemingway and Ranson 2000). The existence of both ACE genes in An. stephensi is approved by other research- ers (Malcolm and Hall 1990, Weill et al. 2002,). But as yet, there is no recorded ACE- based resistance mechanism in An. stephensi (Hemingway and Ranson 2000). It is known that insensitive acetylcholinesterase (AChE) due to a G119S mutation is associated with tolerance to carbamate and organophosphate insecticides in Anopheles gambiae and the mutation can be detected using a PCR-RFLP assay (Weill et al. 2004a). As yet there is no comprehensive research about mechanisms of temephos resistance in An. stephensi in the literature. The current study was designed in order to clarify the enzymatic and molecular mechanisms of temephos resistance in this species. Materials and Methods Study area Eight different areas in two most im- portant malarious provinces of Iran were considered to collect live wild specimens of An. stephensi including: Bandar Abbas Port, Minab County and Hormoodar Village in Hormozgan Province, and Chabahar Port, vil- lages of Bampoor and Abtar from Iranshahr County, villages of Angoori and Machkor from Sarbaz County in Sistan and Baluchi- stan Province (Fig. 1). Mosquito strains The field collected strains of An. stephensi were reared in the insectarum for further tests. A susceptible laboratory strain of An. stephensi (Beech-Lab from insectarium of department of Medical Entomology and Vec- tor Control group, School of Public Health, Tehran University of Medical Sciences) was used to compare the susceptibility status of the field strains. This strain has been main- http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 73 tained in the laboratory without exposure to insecticides for 28 years. Insecticide Technical grade insecticide used in the present study was Temephos 90 % (Batch No: TEM/136-229) which was obtained from Levant Overseas Development Ltd., Argen- teuil, France. Based on pre-tests, five concentrations of the larvicide (0.25, 0.0625, 0.0156, 0.0039 and 0.00195 mg/l) were considered for sus- ceptibility assays. Bioassay consisted of five concentrations resulting 10–90% mortality. Butanone 2% in absolute ethanol was used as a control. Larval bioassays Susceptibility assays was carried out ac- cording to the method described by World Health Organization (WHO 2012). The tox- icity of temephos to An. stephensi, from field-collected population was determined and compared with laboratory reared sus- ceptible Beech-Lab strain. Abbott’s formula was used to correct the observed mortality of larvae. All the data were corrected if the control mortality is between 5 and 20 % (Abbott 1965). Data were analyzed using probit analysis to de- termine the 50% lethal concentration values (LC50) and 90% lethal concentration values (LC90) of the field and Beech-Lab strains (Finney 1971). Selection process The strain which showed the highest re- sistance ratio (RR) to temephos was pre- ceded for selection pressure. This strain was selected for 5 generations by exposing late third or early fourth instars to the concentra- tions which produced 50–70 % mortality (Paeporn et al. 2004). Selection was contin- ued as long as a homogenous resistant pop- ulation with resistance ratio more than 10- fold was achieved. Biochemical assays Thirty mosquito larvae from each suscep- tible and resistant strain were assayed for α- and ß-esterases, mixed function oxidase (MFO) and glutathione-S-transferase (GST), insensitive acetylcholinesterase and PNPA- esterase enzymes. Each larva was homoge- nized in 100 µ L of potassium phosphate (KPO4) buffer (6.6 g dibasic potassium phosphate/1.7g mono basic potassium phos- phate/1000mL distilled water (dH2O), pH 7.2) and then diluted to 2 mL with the same buffer. Each mosquito was analyzed in du- plicate with 100 µ L of mosquito homogenate transferred to two wells on a 96 well flat- bottomed microtitration plate. Absorbance levels were measured spectrophotometrically with a microplate reader (ELX808 Ultra Microplate Reader BIO-TEK ®), at wave lengths indicated for each enzyme, and the mean absorbance calculated based on data for the two replicate wells per mosquito. Procedures were followed based on slight modifications of a protocol from the Centers of Disease Control (Polson et al. 2011). The activities of all enzymes were evaluated ac- cording to this protocol. The details of pro- cedures were described completely in this research (Polson et al. 2011). Reagents and substrates for biochemical assays were pro- vided by Sigma. Data analyses of Biochemical assays Absorbance values which were obtained for mosquito replicates were corrected in relation to the volume of mosquito homoge- nates, the enzyme activity unit and the total protein content of each mosquito (Polson et al. 2011). The means of enzyme activities for each An. stephensi larval strain were com- pared with the susceptible (Beech-Lab strain) by Unpaired t-test, Mann-whitney test (P< 0.05). The Beech-Lab 99th percentile was cal- culated for each enzyme and the percentage of specimens with enzymatic activity above http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 74 that of the Beech-Lab 99th percentile was calculated. Enzyme activities were then clas- sified as “altered”, “incipiently altered” or “unaltered” if the rate was >50 %, between 15 % and 50 % and <15 %, respectively (Montella et al. 2007). Molecular study of resistance Mosquito genomic DNA was extracted from triplex homogenate of mosquitoes by QIAamp DNA Mini Kit. DNA was then PCR amplified with the degenerated primers Moustdir1 5′CCGGGNGCSACYATGTGGAA3′ and Moustrev1 5′ACGATMACGTTCTCYTCCGA3′ accord- ing to the conditions and thermal cycles al- ready introduced by Weill et al. (2004). The PCR products were digested with AluI re- striction enzyme according to the manufac- turer’s instructions and fractionated on a 2% agarose ethidium bromide gel (Weill et al. 2004a). The primers created a 194 bp amplicon in both temephos resistance and susceptible strains, however after restriction enzyme di- gestion, homozygous resistant individuals cut to 120 bp and 74 bp fragments if the G119S mutation was existed. Representative PCR products of both temephos resistance and susceptible strains of An. stephensi were sent for sequencing in order to confirm the PCR-RFLP assays as well as to find other possible mutation on ACE-1 gene except for the G119S mutation. Bioinformatic softwares such as Clustal W2, Blast, and Mega 5 were used for se- quence alignment, homology, and phylogenetic analysis. We also used TranSeq software for translation nucleic acids to amino acids. Results Larval bioassays Considerable variation in temephos re- sistance ratio of filed strains in comparison with susceptible strain was noticed from all the locations studied. A low level of re- sistance ratio was observed in the popula- tions of An. stephensi except in Chabahar strain. (RR= 4.27 folds) compared to Beech- Lab strain (P< 0.05) (Fig. 2). According to our findings, Chabahar strain of An. stephensi with resistance ratio more than 4- folds was chosen for selection process as the most tolerant strain. Selection process After larval bioassays, Chabahar strain was established into the insectary for selec- tion process. Selection process was contin- ued for 15 months. After 5th selection a re- sistant population of An. stephensi was achieved with 15.82 and 35.34-folds re- sistance ratio at LC50 and LC90 level, re- spectively. Biochemical assays Analyses were conducted through com- paring the median value for Beech-Lab strain (S) with those of the temephos se- lected strain (R), for each enzyme. By Un- paired t-test and Mann-Whitney test, the median activity for all enzymes differed sig- nificantly (P< 0.05). According to the classi- fication scheme detailed in method, for each respective enzyme, activities were classified as “unaltered”, “incipiently altered” or “al- tered” if the values were <15%, between15 and 50% and >50%, respectively. Tables 1 and 2 show the number of mosquitoes as- sessed in each assay, along with the median values and percentage of strains with enzy- matic activities in relation to Beech-Lab strain (S). α-Esterase In relation to Beech-Lab strain (S), temephos resistant strain (R) was signifi- cantly different in α-EST activity levels (P< 0.0001). R strain showed altered activity with >50 % (95%) of individuals recording http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 75 activity above that of the 99th percentile of the Beech-Lab reference strain (Table 1). ß-Esterase The median activity levels of ß-EST seen in the resistant strain were significantly dif- ferent from the Beech-Lab strain (P< 0.0001). Based on the classification of ac- tivity profiles, R strain showed unaltered ac- tivity with <15 % (8.33%) of individuals re- cording activity above that of the 99th per- centile of the Beech-Lab reference strain (Table 1). PNPA-Esterase There were significant differences observed between the median PNPA-EST activities of the Beech-Lab strain and temephos resistant strain (P= 0.0096). An unaltered profile of PNPA-EST (6.67%) was found in R strain of An. stephensi (Table 1). Mixed function oxidase (MFO) In relation to Beech-Lab strain (S), temephos resistant strain (R) was significantly different in MFO activity levels (P= 0.0002). R strain showed unaltered activity with <15 % (1.67%) of individuals recording activity above that of the 99th percentile of the Beech-Lab reference strain (Table 2). Glutathione-S-transferase (GST) The GST activity in the Beech-Lab strain was significantly different from that of the temephos resistant strain (P< 0.0001). Based on the classification of activity profiles, R strain showed altered activity with >50 % (86.67%) of individuals recording activity above that of the 99th percentile of the Beech-Lab reference strain (Table 2). Insensitive acetylcholinestersase (iAChE) The rate of activity of AChE in the pres- ence of propoxur for Beech-Lab strain was significantly different from that observed in the temephos resistant strain (P< 0.0001). An altered profile of AChE (90%) was found in the R strain of An. stephensi (Table 2). The activity levels of the enzymes in both strains (R and S) are graphically displayed in scatter plots (Figs. 3–8). Molecular study on ACE-1 Resistance in Anopheles stephensi With Moustdir1 and Moustrev1 primers, a 194 bp amplicon was amplified by PCR. The result of PCR-RFLP with AluI showed that PCR products of both temephos resistance and susceptible strains of An. stephensi were remain intact (Fig. 9) indicating lack of the G119S mutation in ACE-1 of resistance strain. Three specimens from each resistant and susceptible strain of An. stephensi to temephos, were sent for sequencing. The sequences were deposited in the European Nucleotide Archive (ENA) with accession numbers (HG380320- 24). The results of 5 sequencing that were trustable, analyzed with Blast, and Clustal W2 softwares. The Blast analysis revealed that there was no counterpart sequence data of the ACE-1 gene of An. stephensi in the genbank database. The most similar se- quence data available in genbank database were Anopheles albimanus S (Accession number: AJ566402), An. albimanus R (AN: AJ566403) and Anopheles funestus R (AN: DQ534435) (Fig. 10). The results showed sequences of ACE-1 for both resistant and susceptible strains were identical and no G119S mutation was observed in resistance strainthat equenced. The Blast analysis of this region of ACE-1 for An. stephensi, showed sequence of ACE- 1 was more similar to An. albimanus than An. funestus (Table 3). In comparison with available data in genbank, 2 indels, and 34 substitutions were observed (Fig. 10). Con- struction of Phylogram was done using Mega 5 for ACE-1 sequences of this study (An. stephensi R/S) and other available data in genbank (An. albimanus S, An. albimanus R and An. funestus R) (Fig. 11). Amino acids sequences of An. stephensi ACE-1 gene were compared with other similar amino acid sequences of mosquitoes were available in the genbank (Fig. 12). The results showed lack of Glycine to Serine substitu- http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 76 tion at position 119 in the ACE-1 gene that confers high levels of resistance to organ- ophosphate in the resistant (R) strain of An. stephensi of this study. This substitution only observed in R strain of An. albimanus. Two species specific amino acid sequences in ACE-1 gene of An. stephensi were observed in analogy with other sequences. In this spe- cies two Arginine have been substituted with Glutamic acid and Aspartic acid. These dif- ferences seem to be structural and not related to insecticide resistance property of this spe- cies (Fig. 12). Fig. 1. Location of Anopheles stephensi collection sites from malarious areas of Iran, 2011 Fig. 2. Temephos resistance ratio pattern in Anopheles stephensi field strains from malarious area of south- ern Iran Data 1 Mosquito strains nm ol /m g pt n/ m in Be ec h R 0.00000 0.00005 0.00010 0.00015 0.00020 Fig. 3. Activity profile of α-esterase enzymes Data 1 Mosquito strains nm ol /m g pt n/ m in Be ec h R 0.00000 0.00005 0.00010 0.00015 0.00020 0.00025 Fig. 4. Activity profile of ß-esterase enzymes Data 1 Mosquito strains  a bs /m g pt n/ m in Be ec h R -0.05 0.00 0.05 0.10 0.15 Fig. 5. Activity profile of PNPA-esterase enzymes http://jad.tums.ac.ir Published Online: July 16, 2014 Mosquito strains Mosquito strains Mosquito strains Resisrance Ratio ab s/ m g p tn /m in n m ol /m g p tn /m in n m o/ m g p tn /m in J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 77 Table 1. Quantification of enzymatic activity of esterases in two strains (resistant and susceptible) of Anopheles stephensi PNPA-EST(abs/mg ptn/min)ß-EST (nmol/mg ptn/min)α-EST (nmol/mg ptn/min)Strains p99MedianNp99MedianNp99 cMedian bN a 0.079692780.04560881300.000209710.00014011300.000104110.0000688130Beech-Lab %>p99MedianN%>p99MedianN%>p99 dMedianN 6.670.05132526308.330.0001772530950.0001365430Chabahar (Selected with Temephos) a Number of mosquitoes tested. b Median value for each enzymatic activity. c 99th percentile for Beech-Lab reference strain. d Percentage of mosquito specimen with activity above 99th percentile for Beech-Lab reference strain. Table 2. Quantification of enzymatic activity of MFO, GST and iAChE in two strains (resistant and susceptible) of Anopheles stephensi AChE (% activity)GST (nmol/mg ptn/min)MFO (nmol cyt/mg ptn)Strains p99MedianNp99MedianNp99 cMedian bN a 5.365172821.40618583300.00028138-.00085944300.000081080.0000326430Beech-Lab %>p99MedianN%>p99MedianN%>p99 dMedianN 9014.168171183086.670.00033844301.670.0000227930Chabahar (Selected with Temephos) a Number of mosquitoes tested. b Median value for each enzymatic activity. c 99th percentile for Beech-Lab reference strain. d Percentage of mosquito specimen with activity above 99th percentile for Beech-Lab reference strain. Table 3. Blast analyze of ACE-1 region sequence for temephos-resistant Anopheles stephensi R/S (this study) and other species of mosquitoes (Anopheles albimanus S, Anopheles albimanus R and Anopheles funestus R) SeqA Name Length SeqB Name Length Score 1 An. stephensi R/S 160 2 AJ566402 An.albimanus S 162 88.75 1 An. stephensi R/S 160 3 AJ566403 An.albimanus R 162 88.12 1 An. stephensi R/S 160 4 DQ534435 An.funestus R 162 83.12 2 AJ566402 An.albimanus S 162 3 AJ566403 An.albimanus R 162 99.38 2 AJ566402 An.albimanus S 162 4 DQ534435 An.funestus R 162 82.72 3 AJ566403 An.albimanus R 162 4 DQ534435 An.funestus R 162 82.1 http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 78 Data 1 Mosquito strains nm ol c yt /m g pt n Be ec h R 0.00000 0.00002 0.00004 0.00006 0.00008 0.00010 Fig. 6. Activity profile of MFO enzymes Data 1 Mosquito strains nm ol /m g pt n/ m in Be ec h R -0.003 -0.002 -0.001 0.000 0.001 Fig. 7. Activity profile of GST enzymes Data 1 Mosquito strains % r em ai ni ng a ct iv it y Be ec h R -20 0 20 40 60 Fig. 8. Percent remaining activity acetylcholinesterase (AChE) Fig. 9. Diagnostic PCR-RFLP to identify G119S mutation in amplified region of ACE-1 (194 bp) in individuals of Anopheles stephensi. M: 50 bp ladder (Fermentas), Lane 1–6: temephos-resistant strain (Lane 1–2: 4th generation of selected strain with temephos, Lane 3–4: 5th generation of selected strain with temephos, Lane 5–6: 6th generation of selected strain with temephos) CLUSTAL 2.1 multiple sequence alignment Table 4. Summary of some biochemical studies were done in order to characterizing the mechanisms of temephos resistance in different vector species referenceMain mechanism of resistanceinsecticideCountryYearSpecies Peiris and Hemingway 1990 General esterase (α and ß)TemephosSrilanka1990Culex quinquefasciatus Penilla et al. 1998PNPA-estrase and MFOOrganophosphateMexico1998Anopheles albimanus Paeporn et al. 2003General esterase (α and ßTemephosThailand2003Aedes aegypti Saelim et al. 2005General esterase (α and ß)TemephosThailand2005Aedes aegypti Montella et al. 2007General esterase (α and ß), PNPA estrase and GST (only in north-east strain of Brazil) TemephosBrazil2007Aedes aegypti Melo-Santosa et al. 2010General esterase (α and ß) and GSTTemephosBrazil2010Aedes aegypti Polson 2011General esterase (α and ß) , GST, MFO and AChE TemephosTrinidad2011Aedes aegypti This studyα-EST, GST and AChETemephosIran2013Anopheles stephensi http://jad.tums.ac.ir Published Online: July 16, 2014 Mosquito strains Mosquito strains Mosquito strains n m ol /m g p tn /m in n m ol cy t/ m g p tn % r em ai n in g ac ti vi ty J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 79 CLUSTAL 2.1 multiple sequence alignment AJ566402 An.albimanus S TCTCGGAGGACTGCCTGTACATCAACGTGGTGGCGCCGAGGCCACGGCCC 50 AJ566403 An.albimanus R TCTCGGAGGACTGCCTGTACATCAACGTGGTGGCGCCGAGGCCACGGCCC 50 Seq1 TCTCGGAG--ATGTCTGTACATCAACGTGGTAGCACCACGACCCCGTCCC 48 Seq2 TCTCGGAG--ATGTCTGTACATCAACGTGGTAGCACCACGACCCCGTCCC 48 Seq3 TCTCGGAG--ATGTCTGTACATCAACGTGGTAGCACCACGACCCCGTCCC 48 Seq4 TCTCGGAG--ATGTCTGTACATCAACGTGGTAGCACCACGACCCCGTCCC 48 Seq5 TCTCGGAG--ATGTCTGTACATCAACGTGGTAGCACCACGACCCCGTCCC 48 DQ534435 An.funestus R TGTCGGAGGACTGTCTGTACATTAATGTGGTAGCACCACGACCGCGACCG * ****** .** ******** ** *****.**.**..*.** ** ** 50 AJ566402 An.albimanus S AAGAATGCTGCCGTCATGCTGTGGATCTTCGGCGGTGGCTTCTACTCCGG 100 AJ566403 An.albimanus R AAGAATGCTGCCGTCATGCTGTGGATCTTCGGCGGTAGCTTCTACTCCGG 100 Seq1 AAGAATGCTGCCGTTATGCTGTGGATCTTTGGTGGAGGATTCTACTCCGG 98 Seq2 AAGAATGCTGCCGTTATGCTGTGGATCTTTGGTGGAGGATTCTACTCCGG 98 Seq3 AAGAATGCTGCCGTTATGCTGTGGATCTTTGGTGGAGGATTCTACTCCGG 98 Seq4 AAGAATGCTGCCGTTATGCTGTGGATCTTTGGTGGAGGATTCTACTCCGG 98 Seq5 AAGAATGCTGCCGTTATGCTGTGGATCTTTGGTGGAGGATTCTACTCCGG 98 DQ534435 An.funestus R AAGAATGCTGCCGTTATGCTGTGGATCTTTGGCGGTGGATTTTACTCCGG ************** ************** ** **:.*.** ******** 100 AJ566402 An.albimanus S TACGGCCACACTGGACGTGTACGATCACCGGGCGCTCGCCTCGGAAGAGA 150 AJ566403 An.albimanus R TACGGCCACACTGGACGTGTACGATCACCGGGCGCTCGCCTCGGAAGAGA 150 Seq1 TACGGCCACACTGGACGTGTACGATCATCGGGCGCTTGCCTCGGAGGAGA 148 Seq2 TACGGCCACACTGGACGTGTACGATCATCGGGCGCTTGCCTCGGAGGAGA 148 Seq3 TACGGCCACACTGGACGTGTACGATCATCGGGCGCTTGCCTCGGAGGAGA 148 Seq4 TACGGCCACACTGGACGTGTACGATCATCGGGCGCTTGCCTCGGAGGAGA 148 Seq5 TACGGCCACACTGGACGTGTACGATCATCGGGCGCTTGCCTCGGAGGAGA 148 DQ534435 An.funestus R TACCACTACGCTCGACGTGTACGATCACCGTGCGCTCGCATCGGAGGAGA *** .* **.** ************** ** ***** **.*****.**** 150 AJ566402 An.albimanus S ACGTTATCGTAC 162 AJ566403 An.albimanus R ACGTTATCGTAC 162 Seq1 ACGTTATCGTAA 160 Seq2 ACGTTATCGTAA 160 Seq3 ACGTTATCGTAA 160 Seq4 ACGTTATCGTAA 160 Seq5 ACGTTATCGTAA 160 DQ534435 An.funestus R ATGTGATCGTCG * ** *****. 162 Seq1 An. stephensi Chabahar strain with 9.67 resistant ratio, F5 selected with Temephos, ace-1 gene, partial cds Seq2 An. stephensi Chabahar strain with 15.82 resistant ratio,, F6 selected with Temephos, ace-1 gene, partial cds Seq3 An. stephensi Chabahar strain with 6.06 resistant ratio,, F4 selected with Temephos, ace-1 gene, partial cds Seq4 An. stephensi Beech-Lab susceptible strain 5, ace-1 gene, partial cds Seq5 An. stephensi Beech-Lab susceptible strain 9, ace-1 gene, partial cds Fig. 10. Comparison of sequencing results of this study (Seq1, Seq2, Seq3, Seq4, Seq5*) with other three registered genes in gene bank http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 80 Fig. 11. Phylogram a part of ACE-1 for sequences of this study (Anopheles stephensi R/S) and other similar registered genes in gene bank (Anopheles albimanus S, Anopheles albimanus R and Anopheles funestus R) CLUSTAL 2.1 multiple sequence alignment An. albimanus R SEDCLYINVVAPRPRPKNAAVMLWIFGGSFYSGTATLDVYDHRALASEENVIV 53 An. funestus R SEDCLYINVVAPRPRPKNAAVMLWIFGGGFYSGTTTLDVYDHRALASEENVIV 53 An. albimanus S SEDCLYINVVAPRPRPKNAAVMLWIFGGGFYSGTATLDVYDHRALASEENVIV 53 An. stephensi R/S SRRCLYINVVAPRPRPKNAAVMLWIFGGGFYSGTATLDVYDHRALASEENVIV *. *************************.*****:****************** 53  Arginine (R)  Glutamic acid (E)  Aspartic acid (D)  Glycine (G)  Serine (S)  Alanine (A)  Threonine (T) Fig. 12. Comparison of translated ACE-1 region amino acids sequence of Anopheles stephensi R/S (this study) with other similar registered genes in gene bank (Anopheles albimanus S, Anopheles albimanus R and Anopheles funestus R) Discussion In this study, it was found that An. stephensi in southern part of Iran would normally be sus- ceptible to insecticides but become resistant to temephos under insecticide pressure in labor- atory condition. On the other hand, in field con- dition, temephos resistance of this species has been reported from otherneighboring malarious countries such as India and Oman (Vatandoost and Hanafi-Bojd 2005a, Anderasen 2006). This warrants precautions and insecticide vec- tor management (IVM) before the resistance becomes widespread in Iran and neighboring countries. Biochemical assays were carried out by several researchers in order to characteriz- ing the mechanisms of temephos resistance in different vector species. Some of them are mentioned in Table 4. Biochemical assays were done in temephos selected strain of An. stephensi for the first time in the literature. Profile of enzyme ac- tivity in temephos-resistant An. stephensi showed that the mean enzymatic activity of Location of G119S mutation http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 81 α-EST, GST and AChE were classified as altered. These results clarified that metabolic mechanisms have considerable role in re- sistance of An. stephensi to temephos. And the most important mechanisms of resistance are α-EST, GST and AChE. It seems ß-EST, MFO and PNPA-esterase are not important in resistance of An. stephensi to this insecticide. Vector control programs have, for a long time, utilized bioassays to monitor insecti- cide resistance in field mosquito populations. These assays only inform the susceptibility level of a certain population to a specific insecticide (Polson et al. 2011). Biochemical assays also should be included in routine ac- tivities of surveillance programs in order to finding incipiently altered enzyme activity of field populations. These are more informa- tive than the bioassays in that they provide some information on the resistance mecha- nisms involved. With this information we can prevent the development of insecticide resistance in whole population by proper and timely interventions. Biochemical assays should be simultane- ously carried out with routine bioassays in order to improve the surveillance of resistance and monitoring of the efficacy of insecti- cides in malarious area. Considering that the mechanisms and mo- lecular basis of resistance are very diverse, these mechanisms (metabolic and molecular) should be identified as well for each insecti- cide which will be used. In this case, the ef- ficient monitoring strategies shall be appli- cable and finally management of insecticide resistance in vectors can be obtained. High insecticide resistance resulting from insensitive acetylcholinesterase has emerged in mosquitoes. A single mutation (G119S of the ACE-1 gene) explains this high resistance in Culex pipiens and in An. gambiae (Weill et al. 2004 a,b). It has been recently shown that the high insensitivity of acetylcholinesterase displayed by Cx. pipiens and An. gambiae is due to the same glycine to serine substitution (G119S mutation), resulting from a single point mutation GGC to AGC in the gene ACE-1 (Weill et al. 2002). The results of Weill et al. study (2004) showed that, the Gly 119 codon was found serine immutable in 31 vector species in- cluding An. stephensi. Molecular and biochemical assays were carried out to identify ACE-1 mutation in An. gambiae and Culex quinquefasciatus. In this study less than 1 % of mosquitoes showed the presence of the ACE-1 mutation (Corbel et al. 2007). Another study was surveyed acetyl cholinesterase sequencing in Ae. aegypti. In all individuals, a PCR product of 507 bp was amplified. Sequences were aligned and no mutations were observed within this region of ACE. Resistant and susceptible individu- als presented the same nucleotide and amino acid sequence, with 100 % homology to the sequence (Melo-Santosa et al. 2010). These studies clarified that the frequency of ACE-1 mutation into the field population of mosquitoes are very low. The mechanisms of temephos resistance based on the existence of G119S mutation on ACE-1 gene for one of the most important malaria vector An. stephensi were studied. In this study PCR-RFLP showed no G119S mutation was existed in this part of gene of the An. stephensi strains. Resistant and sus- ceptible individuals presented the same nu- cleotide and amino acid sequence, with 100 % homology to the sequence. These results are completely similar to other researchers' results and approved the immutable characteristic of this region of ACE-1 gene in An. stephensi. Finally we can conclude based on molecular studies of temephos resistance, there is no mechanisms of temephos resistance in relation to studied region of Acetylcholinesterase 1 gene in An. stephensi. Probably mechanisms of temephos http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 82 resistance in An. stephensi are more enzymatic or are belong to other parts of the mosquito genome that we didn't studied in this research. Conclusion The results of this study will provide in- formation about mechanisms of temephos resistance in the main malaria vector in Iran. This finding is very crucial for management of malaria vector control. Acknowledgements This article is a part of the first author’s dissertation for fulfillment of a PhD degree in Medical Entomology and Vector Control from Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Teh- ran, Iran. The authors are very grateful to Mr M Yarian from Hormozgan University of Medical Sciences and Mr A Pakari and Mr Shahbakhsh technicians of the National In- stitute of Health Research, Bandar Abbas and Iranshahr Research Stations, for their kind collaboration during this study. This study was financially supported by the Dep- uty for Research, Tehran University of Med- ical Sciences. The authors declare that there is no conflict of interest. References Abbott WS (1965) A method of comparing the effectiveness of an insecticide. J Econ Entomol. 18: 265–267. Anderasen MH (2006) Emerging resistance to temephos in Anopheles stephensi in the Al-Dhahira region of oman. 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