J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … 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 remaiArchivens as a public health prob- the of main malaria SID vector in the Persian Gulf lem in the world. Southern parts of Iran are area (Oshaghi et al. 2006a and 2006b). Pre- involved with this problem (Vatandoost et al. vious studies have shown An. stephensi to be 2010). the most prevalent anopheline species in the Anopheles stephensi is a sub-tropical species malarious areas of southern Iran (Vatandoost et and also an important vector of human ma- al. 2004, Hanafi-Bojd et al. 2012). laria throughout the Middle East and South Temephos, a most widely used organophos- Asian region, including the Indo-Pakistan sub- phorus insecticide, has been included in the continent, with a westward extension through list of World Health Organization (WHO) as Iran and Iraq into the Middle East and Ara- a suitable and safe mosquito larvicide that bian Peninsula. This species is considered to be can be used even in drinking water for con- *Corresponding author: Prof Hassan Vatandoost, E- 71 mail: [email protected], Dr Mohammad Ali http://jad.tums.ac.ir www.SID.ir Oshaghi, E-mail: [email protected] Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … trolling of the most mosquito vectors .The aegypti and An. stephensi, both of these genes toxicity of this insecticide is low and unlike- are also sex linked (Hemingway and Ranson ly to present acute hazard for human (WHO 2000). The existence of both ACE genes in 2006). An. stephensi is approved by other research- Temephos (EC 50%) has been used for ers (Malcolm and Hall 1990, Weill et al. some years for larval control program of 2002,). But as yet, there is no recorded ACE- malaria in Southern Iran (Vatandoost et al. based resistance mechanism in An. stephensi 2006). Many studies on the susceptibility (Hemingway and Ranson 2000). It is known level of An. stephensi to various pesticides that insensitive acetylcholinesterase (AChE) have been done in Iran and other countries. due to a G119S mutation is associated with Resistance of An. stephensi to different insec- tolerance to carbamate and organophosphate ticide was reported from around the world insecticides in Anopheles gambiae and the (Vatandoost et al. 1996). Different levels of mutation can be detected using a PCR-RFLP resistance to larvicides were reported in assay (Weill et al. 2004a). anopheline malaria vectors worldwide. Anoph- As yet there is no comprehensive research eles stephensi has an extensive resistance com- about mechanisms of temephos resistance in paring to other species and is resistant or tol- An. stephensi in the literature. The current erant to fenitrothion, temephos and fenthion study was designed in order to clarify the in India, fenitrothion and pirimiphos-methyl enzymatic and molecular mechanisms of in Iraq, fenitrothion, pirimiphos-methyl, temephos resistance in this species. chlorfoxim and foxim in Iran and fenitrothion in Pakistan (Vatandoost and Hanafi-Bojd 2005a). Resistance of other anopheline mos- Materials and Methods quito such as An. dthali to temephos also was reported (Hanafi-Bojd et al. 2006). Study area In 2006 for the first time in the Middle Eight different areas in two most im- East, resistance to temephos was confirmed portant malarious provinces of Iran were in An. stephensi breeding in water storage considered to collect live wild specimens of tanks in the Al-Dhahira region of Oman An. stephensi including: Bandar Abbas Port, (Anderasen 2006). The level of resistance Minab County and Hormoodar Village in was 2.5 times higher than that of the WHO Hormozgan Province, and Chabahar Port, vil- diagnostic dose (0. 25 mg/l). However, there lages of Bampoor and Abtar from Iranshahr was no confirmed report of resistance of An. County, villages of Angoori and Machkor stephensi to temephos in Iran. Previous stud- from Sarbaz County in Sistan and Baluchi- ies in Iran showed thatArchive this species was com- stan of Province SID(Fig. 1). pletely susceptible to temephos at the WHO diagnostic dose (Vatandoost et al. 2004, Mosquito strains Vatandoost and Hanafi-Bojd 2005a, Vatandoost The field collected strains of An. stephensi et al. 2005b, Vatandoost et al. 2006). were reared in the insectarum for further tests. One of the most important molecular mech- A susceptible laboratory strain of An. anisms of resistance to organophosphate in- stephensi (Beech-Lab from insectarium of secticide in mosquitoes is structural muta- department of Medical Entomology and Vec- tions that occur in acetylcholinesterase gene. tor Control group, School of Public Health, In mosquitoes two cholinesterase genes are Tehran University of Medical Sciences) was existed (ACE-2 and ACE-1). ACE genes used to compare the susceptibility status of have been cloned from the mosquitoes Aedes the field strains. This strain has been main- 72 http://jad.tums.ac.ir www.SID.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 71–83 A Soltani et al.: Resistance Mechanisms of … tained in the laboratory without exposure to Biochemical assays insecticides for 28 years. Thirty mosquito larvae from each suscep- tible and resistant strain were assayed for α- Insecticide and ß-esterases, mixed function oxidase Technical grade insecticide used in the (MFO) and glutathione-S-transferase (GST), present study was Temephos 90 % (Batch insensitive acetylcholinesterase and PNPA- No: TEM/136-229) which was obtained from esterase enzymes. Each larva was homoge- Levant Overseas Development Ltd., Argen- nized in 100 µL of potassium phosphate teuil, France. (KPO4) buffer (6.6 g dibasic potassium Based on pre-tests, five concentrations of phosphate/1.7g mono basic potassium phos- the larvicide (0.25, 0.0625, 0.0156, 0.0039 phate/1000mL distilled water (dH2O), pH and 0.00195 mg/l) were considered for sus- 7.2) and then diluted to 2 mL with the same ceptibility assays. Bioassay consisted of five buffer. Each mosquito was analyzed in du- concentrations resulting 10–90% mortality. plicate with 100 µL of mosquito homogenate Butanone 2% in absolute ethanol was used transferred to two wells on a 96 well flat- as a control. bottomed microtitration plate. Absorbance levels were measured spectrophotometrically Larval bioassays with a microplate reader (ELX808 Ultra Susceptibility assays was carried out ac- Microplate Reader BIO-TEK ®), at wave cording to the method described by World lengths indicated for each enzyme, and the Health Organization (WHO 2012). The tox- mean absorbance calculated based on data icity of temephos to An. stephensi, from for the two replicate wells per mosquito. field-collected population was determined Procedures were followed based on slight and compared with laboratory reared sus- modifications of a protocol from the Centers ceptible Beech-Lab strain. of Disease Control (Polson et al. 2011). The Abbott’s formula was used to correct the activities of all enzymes were evaluated ac- observed mortality of larvae. All the data cording to this protocol. The details of pro- were corrected if the control mortality is cedures were described completely in this between 5 and 20 % (Abbott 1965). Data research (Polson et al. 2011). Reagents and were analyzed using probit analysis to de- substrates for biochemical assays were pro- termine the 50% lethal concentration values vided by Sigma. (LC50) and 90% lethal concentration values (LC90) of the field and Beech-Lab strains Data analyses of Biochemical assays (Finney 1971). Absorbance values which were obtained Archivefor of mosquito SIDreplicates were corrected in Selection process relation to the volume of mosquito homoge- The strain which showed the highest re- nates, the enzyme activity unit and the total sistance ratio (RR) to temephos was pre- protein content of each mosquito (Polson et ceded for selection pressure.