J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 147 Original Article Chemical Composition, Larvicidal and Repellency Properties of Cionura erecta (L.) Griseb. Against Malaria Vector, Anopheles stephensi Liston (Diptera: Culicidae) Ehssan Mozaffari 1, Mohammad Reza Abai 1, Mahnaz Khanavi 2, *Hassan Vatandoost 1, Mohammad Mehdi Sedaghat 1, Abbas Moridnia 1, Mahsa Saber-Navaei 2, Alireza Sanei- Dehkordi 1, Fatemeh Rafi 1 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (Received 30 June 2013; accepted 1 Oct 2013) Abstract Background: Application of plant derivatives have been suggested as alternative sources for mosquito control. Methods: The root essential oil and methanol extract of Cionura erecta (L.) Griseb was tested under laboratory conditions for larvicidal and skin repelleny activities against Anopheles stephensi. The chemical compositions of essential oils were analyzed using gas chromatography- mass spectrometry. Results: Among the five concentrations tested, the 320 ppm of essential oil and 1280 ppm of methanolic extract had the most toxic effects yielding 100% mortality. The LC50 values of C. erecta for both essential oil and methanolic extract were 77.30 and 250.38 ppm, respectively. A total of 19 compounds were identified in essential oil of root. The major components were detected in root essential oil including Cedren-9-one (7.89%), alpha cadinol (5.67%), eugenol (4.02%) and alpha muurolene (3.58%). The protection time of 50% solution of essential oil against bites of An. stephensi was 2.28 hour on white rabbit and the ED50 and ED90 values of the essential oil were 10.12 and 23.01 ppm respectively. Conclusion: The findings suggest that C. erecta oil has a potential source as larvicidal and repellency properties against An.stephensi. Keywords: Cionura erecta, Extract, Essential Oil , Anopheles stephensi, Larvicidal Effect, Repellency Introduction The mosquitoes are the important vectors of human diseases and can be transmissed ma- laria, dengue fever, yellow fever and filariasis. They also bother the people both inside and outside places (Lehane 1991). Malaria is one of arthropod-borne disease and approximately 83000 deaths are reported annually in the world (WHO 2012). At the present, this para- sitic disease is one of the main health problems in Iran. Several chemical compounds have been used against malaria vectors as larvicides including organophosphates, insect growth regulator and microbial dervied compounds (Ghosh et al. 2012). Use of synthetic insecticides is causing various problem such as environmental pollu- tion, insecticide resistance and toxic hazards to humans and animals (Aktar et al. 2009, Kunz and Kemp 1994, Vatandoost et al. 2005). Therefore using plant derivatives have been suggested as alternative sources for mosquito control. They are selective, safe and biode- grade to break down readily in soil and are not stored in plant or animal tissue (Isman 2000, 2006). The various extracts of local plants have been investigated against An. *Corresponding author: Prof Hassan Vatandoost, Email: hvatandoost1@yahoo.com http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 148 stephensi (Hadjiakhoondi et al. 2000, 2003, 2005, Vatandoost and Vaziri 2004, 2012, Se- daghat et al. 2010, 2011a,b, Vatandoost et al. 2012, Khanavi et al. 2013). Personal protection using repellents is one of the effective methods for preventing mos- quito-borne diseases by reducing man–mos- quito contact. The most common insect repel- lent is DEET (N, N-Diethyl-m-toluamide) moreover, there are several reports about of its toxicity against the skin, nervous and im- mune systems (Fradin et al. 1998, Katz et al. 2008, Nerio et al. 2010). Insect repellents from natural sources are a good and safe approach for personal protection against the mosquito bites (Fradin et al. 1998). The repellent effect of the plant essential oil has been examined against mosquito species in Iran (Oshaghi et al. 2003, Yaghoobi-Ershadi et al. 2006, Vatandoost and Hanafi-Bojd. 2008, Tavassoli et al. 2011). Cionura erecta is a woody-based plants 50–100cm high and up to 2m wide perennial with numerous herbaceous rambling stems, often twining at the tips, with copious poison- ous milky juice and is widespread in the Medi- terranean region which is classified in the fam- ily Apocynaceae. This plant known as poison- ous composition which traditionally used for killing pest animals, therefore we used of it. Previous studies indicated that safranal, (Z)-3- hexenyl benzoate, heneicosane were obtained as major components from the essential oil of C. erecta in Turkey (Myrianthopoulos et al. 2007). This study was aimed at assessing the po- tential of plant essential oil and methanolic extract for possible use as larvicidal or repel- lency activites against An. stephensi under laboratory conditions and to determine the chemical composition of the essential oil. Materials and Methods Mosquito rearing The tested mosquitoes were the colony of An. stephensi which obtained from the Insec- tary of School of Public Health, Tehran Uni- versity of Medical Sciences, Iran, and main- tained at 29±1 ºC with a photoperiod of 12 hours light and 12 hours dark in 60±10% rel- ative humidity. The enriched wheat germ was used as food source. Larvae of An. stephensi were continuously available for the larvicidal and repellency experiments. Starved 7 to 10 days old females were used for the repel- lency tests and the early fourth-instars larvae used for the larval bioassays. Plant materials The fresh branch and root of C. erecta col- lected in August 2011 from rural areas located in western part of Ilam Province, Iran (33º 46' N, 46º 11' E at elevation 1195m) (Fig. 1). The specimens was identified and authenti- cated by the Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences. Essential oil isolation Fresh roots (500g) of C. erecta were sub- jected to hydrodistillation using a modified Clevenger-type (Pyrexfan®) apparatus for 3 hour, the oil obtained was separated from water and dried over anhydrous Na2SO4 and transferred into airtight vials at 5 °C. Analysis of essential oils Chemical composition of C. erecta was an- alyzed using an Agilent 7890–5975 gas chro- matography-mass spectrometer. With a HP- 5MS (5% Phenyl Methyl Silox) capillary col- umn (30m×0.25mm, film thickness 0.25μm), split ratio, 1: 1, and using a flame ionization de- tector. The GC was programmed at 50 °C for 2min and then increased at 5 °C/min to 280 °C, and finally held with an isothermal for 3min. The injector temperature was 280 °C. The flow rate of the carrier gas was 1ml/min. The iden- tification of compounds was performed by comparing their retention times and mass spectra with mass spectra from Wiley library. Additional identification was achieved by comparing linear retention indices, relative to n-alkanes, to those from literature (Adams http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 149 2001). Details on the identification of volatile compounds were reported in previous paper (Myrianthopoulos et al. 2007). Methanolic extract of plant The branch and root of C. erecta were air- dried at room temperature and 100g of plant were submitted to percolation separately with methanol (80%) during 3 days, and this pro- cedure were repeated for three times succes- sive and totally last nine day at laboratory tem- perature (22 to 25 °C). The extracts were next evaporated in a rotary evaporator (Heidolph Persia®). Larvicidal tests The essential oil and methanol extract first dissolved in absolute ethanol (99.0%) and methanol (99.0%) respectively. The 400ml glass beakers were used for the treatment or untreated experiments. The third or early fourth instar larvae were exposed to 10, 20, 40, 80 and 160ppm and 40, 80, 160, 320, 640, 1280 ppm of both essential oil and methanolic ex- tract respectively according to standard WHO procedure (WHO 1981). Repellency tests The white rabbits (Oryctolagus cuniculus) (laboratory reared albino male aged six months) were used to determine both protection time and effective dosage. The 25, 50 percent and pure essential oil of C. erecta was prepared using absolute ethanol as well as this solvent used for untreated group against An. stephensi on the shaved back of male rabbits with 4 repitations. The procedure for determination of effective dosages of the repellents was adopted by the standard method of American Society for Testing and Material (ASTM 2000). The testing kit was made of plexiglas cube at dimension of 4×5×18cm having four rec- tangular holes 2 ×3cm. Before starting the test for determination of effective dosage, the ab- domen skins of rabbits were cleaned with al- cohol and the kit was fixed on the abdomen. The eligibility of the rabbits for repellency tests was 10 landings or probes within 30 seconds. Each of 4 adjacent cells of kit was provided with 5 female 7–10 days mosqui- toes that randomly selected from a cage con- taining 150 starved mosquitoes. Five circles were drawn on the rabbit's skin. The drawn circles on the abodemen skin’s of holded rabbit were treated with 25µ l of essential oil diluted with absolute alcohol at 2, 4, 8, 16, 32ppm with 4 repitations. The serial dilu- tions were applied on 3 holes as well as the absolute ethanol was applied in remaining con- trol circle. The treated circles were allowed to dry, and then test apparatus containing starved mosquitoes were fixed on the treated skin. The counts of probing and biting were rec- orded at 1 minute intervals up to 5 minutes. After each test, the mosquitoes were trans- ferred to netted cups and the mortality of mosquitoes was recorded after 24 hours .The ED50 and ED90 values and regression param- eters were analyzed using probit 79 program and the regression lines were plotted in Microsoft Excel 2007. Ethical approval Animal experiments were performed after obtaining Institutional Animal Ethical Com- mittee’s approval from Tehran University of Medical Sciences. Results GC-mass analysis The hydrodistillation of the C. erecta root gave oil in 0.16% (w/w) yield on fresh weight material. A total of 19 compounds was 36.4% in roots of C. erecta were identified (Table 1). The major components in root oil were cedren- 9-one (7.89%), alpha cadinol (5.67%), eugenol (4.02%) and alpha muurolene (3.58%) respec- tively. Mosquito larvicidal activity The larvicidal activities of both essential http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 150 oil and methanol extract of the C. erecta root against An. stephensi larvae under laboratory conditions are shown in Table 2. Among the five concentrations tested, the dosages of 320 ppm and 1280ppm of essential oil and meth- anolic extract were respectively found to be the most toxic with 100% larval mortality. The essential oil C. erecta extracted with root and showed the higher toxicity than methanolic extract against the larvae. The LC50 and LC90 values of C. erecta essential oil were 77.30 and 199.58ppm, and for methanolic extract were recorded 250.38 and 490.00ppm, respectively. Effective doses The ED50 and ED90 values of C. erecta essential oil were 10.12 and 23.01 ppm with confidence intervals ranged, 7.89–13.9 and 16.12–50.37 respectively (Table 4). Protection time The 25, 50 percent and pure essential oil C. erecta against An. stephensi on animal subject were provided 2.0–3.15 hours protec- tion. The repellent failure time was ranged 2.5–4.25 hours (Table 3). Effective doses The ED50 and ED90 values of C. erecta essential oil were 10.12 and 23.01ppm with confidence interval ranged, 7.89–13.9 and 16.12–50.37 respectively (Table 4). Protection time The 25%, 50% and 100% essential oil C. erecta against An. stephensi on animal sub- ject were provided 2.0–3.15 hours protection. The repellent failure time was ranged 2.5– 4.25 hours (Table 3). Table 1. Chemical constituents of root essential oil from Cionura erecta No. Compounds Composition% RI 1 2,4 decadienal 1.3 1307 2 dimethyl phenyl acetate 1.25 1315 3 Eugenol 4.02 1360 4 beta elemen 0.332 1391 5 alpha humulene 0.21 1457 6 Trans caryophyllene 0.71 1473 7 alpha muurolene 3.58 1483 8 delta cadinene 1.52 1404 9 caryophyllene oxide 1.38 1540 10 Viridiflorol 0.55 1546 11 Silphiperfolenone 0.85 1551 12 Trans cadinene 0.64 1569 13 alpha cadinol 5.67 1577 14 Eudesmol 1.78 1584 15 gama epoxy elemen 0.69 1598 16 Cedren-9-one 7.89 1633 17 Isolongifolene-5-one 2.15 1644 18 Tetradecanol 1.34 1648 19 Cadalene 0.58 1677 36.44 *RI: Retention indices determined on HP-5 column http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 151 Fig. 1. The plant C. erecta in its natral habitat, Ilam Provinc, west of Iran (original) Table 2. LC50 and LC90 values of essential oil and methanolic extract of Cionura erecta roots against larvae of An. stephensi Type of extraction a b ± SE LC50 (ppm) ± 95%C.L. LC90 (ppm) ± 95%C.L. χ2 (heterogeneity) χ2 table (df) p-Value Essential oil -5.87 10.11 ± 0.34 69.28 77.30 86.40 169.71 199.58 244.91 5.419 * 11.345 (3) 0.01 Methanol extract 4.40 10.54 ± 0.34 229.078 250.38 273.66 434.03 490.00 571.00 11.999 * 13.277 (4) 0.01 * No heterogeneity Table 3. Protection and failure times of essential oil of Cionura erecta against Anopheles stephensi on abdomen of albino rabbits at laboratory condition Concentration of essential oil Protection time (h)± SD Failure time (h)±SD 25% 2.01±.95 2.5±.5 50% 2.28±15.6 3.25±17 100% 3.15±17.3 4.25±17.3 Tables 4. Effective doses of essential oils Cionura erecta (L.) roots against An. stephensi on albino rabbits a b ± SE ED50 ( mg/cm 2) ± 95%C.L. ED90 ( mg/cm 2) ± 95%C.L. χ2 (heterogeneity) χ2 table (df) p-Value -3.59 3.575 ± 0.784 7.89 0.01012 13.89 16.12 0.02301 50.37 3.048* 13.277 (3) 0.01 * No heterogeneity Discussion Application of larvicides and repellents are generally accepted as the playing a significant role in control of the mosquitoes. In this study, major constituents of root essential oil of C. erecta were evaluated. Cedren-9-one (7.89%), alpha cadinol (5.67 %), eugenol (4.02%) and alpha muurolene (3.58%) were found as main compounds. The chemical ingredients of C. erecta essen- tial oil was reported comprised 72 compo- http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 152 nents, from which the main one considered as safranal (16.8%), (Z)-3-hexenyl benzoate (6.1%), heneicosane (5.7%) linalool (4.8%) and tricosane (4.4%) (Myrianthopoulos et al. 2007). Some constitute was not found in our study. According to the larvicidal assay, the es- sential oil and methanol extract of C. erecta were effective against An. stephensi with LC50 and LC90 values of 77.30ppm and 250.38 ppm, respectively. The bioassay of different herbal extracts has been stidied against An. stephensi larvae in Iran. There is a report about the efficacy of the essential oil and methanolic extract of Eucalyptus camaldulensis against An. stephensi in which, the LC50 and LC90 values were found 89.85ppm and 397.75 ppm, respectively (Sedaghat et al. 2010). The larvicidal activity of Azadirachta indica extract against An. stephensi were gained 0.35 ppm and 1.81ppm respectively for LC50 and LC90 values (Vatandoost and Vaziri 2004). Also the LC50 and LC90 of Cupressus arizonica essential oil have been reported respectively 79.30ppm and 238.89ppm against An. stephensi (Sedaghat et al. 2011b). The larvicidal activ- ity of three plants from the family Apiaceae have been studied and the LC50 values of three essential oils ranged from 20.10 to 120.95ppm (Sedaghat et al. 2011a). In the other study, the efficacy of Kelussia odoratissima essen- tial oil was evaluated at dose of 10ppm induced 100% larval mortality, against larvae of both An. stephensi and Cx. pipiens (Vatandoost et al. 2012). The repellency effect of the C. erecta es- sential oil against An. stephensi was first eval- uated under laboratory conditions. The mean protection time of 50% essential oil of C. erecta provided 2.15 hours protection against An. stephensi. The figures for for ED50 and ED90 values were 10.12 and 23.01ppm respectively. The repellency effect of essential oils of many plants has been evaluated against An. stephensi (Ansari et al. 2000, Prajapati et al. 2005, Klun et al. 2006, Rajkumar and Jebanesan 2007, Mullai et al. 2008, Pandey et al. 2009, Govindarajan et al. 2011, Prabhu et al. 2011). The repellency effect of essential oils of both Myrtus communis and Calendula officinalis had been reported and the ED50 values were 0.11 and 0.6 mg/cm2, respectively on human subjects (Tavassoli et al. 2011). Other labor- atory trial revealed the repellency of 3 chem- ical and herbal repellents against An. stephensi. The ED50 value of neem tree’s essential oil was 0.191 mg/cm2 against field strain of mos- quitoes (Vatandoost et al. 2008). The results indicated both the repellency of essential oil as well as the larvicidal activity of C. erecta extract against An. stephensi. Conclusion Results of this study will provide a clue for possible use of plants for control of mosquito-borne disease. Acknowledgments This study was funded and supported by Tehran University of Medical Sciences (TUMS); Grant no. 90-04-27-16617. We are thankful to the staff of Insectary of Culicidae, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences for the mass production of the mosquitoes for this study. The authors declare that there is no conflict of interests. References Adams RP (2001) Identification of essential oil components by gas chromatography/ mass spectroscopy. Allured, Carol Stream IL, USA. Ansari MA, Vasudevan P, Tandon M, Razdan RK (2000) Larvicidal and mosquito re- pellent action of peppermint (Mentha piperita) oil. Bioresour Technol. 71: 267–271. http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 153 Aktar MDW, Sengupta D, Chowdhury A (2009) Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip Toxicol. 2(1): 1–12. ASTM (2000) Standard test methods for laboratory testing of non-commercial mosquito repellent formulation on the skin. American Society for Testing and Materials, ASTM-E951-94 (Reapproved 2000). Fradin MS (1998) Mosquitoes and mosquito repellents. Ann of Internal Med. 128: 931–940. Ghosh A, Chowdhury N, Chandra G (2012) Plant extracts as potential mosquito larvicides. Indian J Med Res. 135(5): 581–598. Govindarajan M, Mathivanan T, Elumalai K, Krishnappa K, Anandan A (2011) Ovi- cidal and repellent activities of botan- ical extracts against Culex quinque- fasciatus, Aedes aegypti and Anopheles stephensi (Diptera: Culicidae). Asian Pac J Trop Biomed. 1: 43–48. Hadjiakhoondi A, Vatandoost H, Abousaber M, Khanavi M, Abdi L (2000) Chemi- cal composition of the essential oil of Tagetes minuta L. and its effects on Anopheles stephensi larvae in Iran. J Med Plants. 7: 33–100. Hadjiakhoondi A, Vatandoost H, Jamshidi A, Bagherj Amiri E (2003) Chemical con- stituents and efficacy of Cymbopogon olivieri (Boiss) bar essential oil against malaria vector, Anopheles stephensi. Daru. 11: 125–128. Hadjiakhoondi A, Vatandoost H, Khanavi M, Abai MR (2005) Biochemical investiga- tion of different extracts and larvicidal activity of Tagetes minuta L on Anoph- eles stephensi larvae. Iran J Pharm Sci. 1: 81–84. Isman MB (2000) Plant essential oils for pest and disease management. Crop Prot. 19: 603–608. Isman MB (2006) Botanical insecticides, de terrents, and repellents in modern agri- culture and an increasingly regulated world. Annu Rev Entomol. 51: 45–66. Katz TM, Miller JH, Hebert AA (2008) Insect repellents: historical perspectives and new developments. J Am Acad Der- matol. 58: 865–871. Khanavi M, Vatandoost H, Khosravi-Dehaghi N, Sanei-Dehkordi A, Sedaghat MM, Hadjiakhoondi A, Hadjiakhoondi F (2013) Larvicidal activities of some Iranian native plants against the main malaria vector, Anopheles stephensi. Acta Med Iran. 51: 141–147. Klun JA, Khrimian A, Debboun M (2006) Re- pellent and deterrent effects of SS220, Picaridin, and Deet suppress human blood feeding by Aedes aegypti, Anoph- eles stephensi, and Phlebotomus papa- tasi. J Med Entomol. 43: 34–39. Kunz SE, Kemp DH (1994) Insecticides and acaricides: resistance and environmen- tal impact. Rev Sci Tech. 13: 1249. Lehane MJ (1991) Biology of blood-sucking insects. London, UK: Harper Collins Academic. Mullai K, Jebanesan A, Pushpanathan T (2008) Mosquitocidal and repellent activity of the leaf extract of Citrullus vulgaris (cu- curbitaceae) against the malarial vec- tor, Anopheles stephensi liston (diptera culicidae). Eur Rev Med Pharmacol Sci. 12: 1–7. Myrianthopoulos V, Fokialakis N, Melliou E, Mitaku S (2007) Chemical composi- tion of essential oil of Cionura Erecta (Asclepiadaceae) Inforescences. J Essent Oil Res. 19: 266–268. Nerio LS, Olivero-Verbel J, Stashenko E (2010) Repellent activity of essential oils: a review. Bioresour Technol. 101: 372–378. Oshaghi MA, Ghalandari R, Vatandoost H, Shaeghi M, Kmali-Nejad M, Tourabi- Khaledi H, Abolhassani M, Hashem- zadeh M (2003) Repellent effect of ex- http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 154 tracts and essential oil of Citrus limon (Rutaceae) and Melissa officinalis (Labiatae) against main malaria vector, Anopheles stephensi (Diptera: Culicidae) in Iran. Iran J Public Health. 32: 47–52. Pandey SK, Upadhyay S, Tripathi AK (2009) Insecticidal and repellent activities of thymol from the essential oil of Trachy- spermum ammi (Linn) Sprague seeds against Anopheles stephensi. Parasitol Res. 105: 507–512. Prabhu K, Murugan K, Nareshkumar A, Ramasubramanian N, Bragadeeswaran S (2011) Larvicidal and repellent po- tential of Moringa oleifera against ma- larial vector, Anopheles stephensi Lis- ton (Insecta: Diptera: Culicidae). Asian Pac J Trop Biomed. 1: 124–129. Prajapati V, Tripathi AK, Aggarwal KK, Khanuja SPS (2005) Insecticidal, re- pellent and oviposition-deterrent activ- ity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. Bioresour Tech- nol. 96: 1749–1757. Rajkumar S, Jebanesan A (2007) Repellent activity of selected plant essential oils against the malarial fever mosquito Anopheles stephensi. Trop Biomed. 24: 71–75. Sedaghat MM, Sanei-Dehkordi A, Abai MR, Khanavi M, Mohtarami F, Saim abadi Y, Vatandoost H (2011a) Larvicidal Activity of Essential Oils of Apiaceae Plants against Malaria Vector, Anoph- eles stephensi. Iran J Arthropod-Borne Dis. 5(2): 51–59. Sedaghat MM, Sanei-Dehkordi A, Khanavi M, Abai MR, Hadjiakhoondi A, Mohtarami F, Vatandoost H (2010) Phytochemistry and larvicidal activity of Eucalyptus camaldulensis against malaria vector, Anopheles stephensi. Asian Pac J Trop med. 3: 841–845. Sedaghat MM, Sanei-Dehkordi A, Khanavi M, Abai MR, Mohtarami F, Vatan- doost H (2011b) Chemical composi- tion and larvicidal activity of essential oil of Cupressus arizonica E.L. Greene against malaria vector Anopheles ste- phensi Liston (Diptera: Culicidae). Phar- macognosy Res. 2: 135–139. Tavassoli M, Shayeghi M, Vatandoost H, Khoobdel M, Salari M, Ghaderi A, Rafi F (2011) Repellency Effects of Essen- tial Oils of Myrtle (Myrtus communis), Marigold (Calendula officinalis) Com- pared with DEET against Anopheles stephensi on Human Volunteers. Iran J Arthropod-Borne Dis. 5: 10–22. Vatandoost H, Hanafi-Bojd AA (2005) Current resistant status of Anopheles stephensi Liston to different larvicides in Hor- mozgan Province, southeastern Iran. Pak J Biol Sci. 8: 1568–1570. Vatandoost H, Hanafi-Bojd AA (2008) La- boratory evaluation of 3 repellents against Anopheles stephensi in the Is- lamic Republic of Iran. East Mediterr Health J. 14: 260–267. Vatandoost H, Mashayekhi M, Abai MR, Aflatoonian MR, Hanafi-Bojd AA, Sharifi I (2005) Monitoring of insecti- cides resistance in main malaria vectors in a malarious area of Kahnooj district, Kerman Province, southeastern Iran. J Vector Borne Dis. 42: 100–108. Vatandoost H, Sanei Dehkordi A, Sadeghi SMT, Davari B, Karimian F, Abai MR, Sedaghat MM (2012) Identification of chemical constituents and Larvicidal activity of Kelussia odoratissima Mozaffarian essential oil against two mosquito vectors Anopheles stephensi and Culex pipiens (Diptera: Culicidae). Exp Parasitol. 132: 470–474. Vatandoost H, Vaziri VM (2004) Larvicidal activity of a neem tree extract (Neemarin) against mosquito larvae in the Islamic Republic of Iran. East Mediterr Health J. 10: 573–581. Vatandoost H, Zahirnia AH (2010) Respon- http://jad.tums.ac.ir Published Online: April 09, 2014 J Arthropod-Borne Dis, December 2014, 8(2): 147–155 E Mozaffari et al.: Chemical Composition … 155 siveness of Anopheles maculipennis to different imagicides during resurgent malaria. Asian Pacific J Trop Med. 3: 360–363. WHO (1981) Instruction for determining the susceptibility or resistance of mosquito larvae to insecticides. World Health Organization-VBC. WHO (2012) World Malaria Report. World Health Organization. Yaghoobi-Ershadi MR, Akhavan AA, Jahani- fard E, Vatandoost H, Amin GH, Moosavi L, Ramazani AR, Abdoli H, Arandian MH (2006) Repellency effect of Myrtle essential oil and DEET against Phlebo- tomus papatasi, under Laboratory Con- ditions. Iran J Public Health. 35: 7–13. http://jad.tums.ac.ir Published Online: April 09, 2014