J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

Original Article Chemical Composition, Larvicidal and Repellent Properties of erecta (L.) Griseb. Against Malaria Vector, Anopheles stephensi Liston (Diptera: Culicidae) Under Laboratory Conditions

Ehssan Mozaffari 1, Mohammad Reza Abai 1, Mahnaz Khanavi 2, *Hassan Vatandoost 1, Mohammad Mehdi Sedaghat 1, Alireza Sanei-Dehkordi 1, Abbas Moridnia 1, Mahsa Saber- Navaei 2, 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: the using of derivatives have been suggested as alternative sources for mosquito control. Methods: The root essential oil and methanol extract of Cionura erecta (L.) Griseb was examined under laboratory conditions for larvicidal and skin repellent activities against Anopheles stephensi. The chemical compositions of essential oils were analyzed using gas chromatography- mass spectrometry. Results: Among the five concentrations tested, the dosage of 320ppm of essential oil and 1280 ppm of methanol extract had the most toxic effect yielding 100% mortality. The LC50 values of C. erecta for both essential oil and methanol extract were 77.30 and 250.38ppm, respectively. A total of 19 compounds amounting to 37% in roots of C. erecta were identified. The major components in root oil were 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 from C. erecta against bites of An. stephensi on white rabbit was 2.28 hour and also the ED50 and ED90 value of the C. erecta essential oil were 10.12 and 23.01ppm respectively. Conclusion: The findings suggest that C. erecta oil is a potential source of larvicidal and repellent compounds.

Keywords: Malaria, Cionura erecta, Vector, Anopheles stephensi, repellents- larvicides and plant derivatives

Introduction

The mosquitoes (family Culicidae) are im- At present, several chemical compounds portant in medical entomology research be- have been used against malaria vectors in- cause can threaten the human health by trans- clude organophosphates, insect growth regu- mitting diseases of malaria, dengue fever, yel- lator and microbial larvicides (Coluzzi 1992). low fever and filariasis, They are also causa- Use of synthetic insecticides is causing vari- tive agents for nuisance of human in both in- ous problem such as environmental pollution, side and outside places (Lehane 1991). Malar- insecticide resistance and toxic hazards to ia is the most important one among arthropod- humans and animals (Mellanby 1967, WHO borne diseases and responsible for approxi- 1985, Kunz and Kemp 1994, Alloway and mately one million deaths annually and has af- Ayres 1997, Vatandoost et al. 2005, Davari fected nearly half of the world’s population et al. 2006, 2007). (WHO 2008). At the present, this parasitic dis- Therefore using plant derivatives have been ease is one of the main health problems in Iran. suggested as alternative sources for mosquito

*Corresponding author: Dr Hassan Vatandoost, Email: [email protected] J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

control. They are selective, safe and biode- conditions and to determine the chemical com- grade to break down readily in soil and are position of the essential oil. not stored in plant or animal tissue (Isman 2000, 2006). The various extracts of local Materials and Methods have been investigated against An. stephensi (Hadjiakhoondi et al. 2000, 2003, Mosquito rearing 2005, Vatandoost and Vaziri 2004, 2012, Se- The tested mosquitoes were the colony of daghat et al. 2010, 2011a,b, Vatandoost et al. An. stephensi which obtained from the Insec- 2012, Khanavi et al. 2013). tary of School of Public Health, Tehran Uni- Personal protection using repellents is one versity of Medical Sciences, Iran, and main- of the effective methods for preventing mos- tained at 27 ºC with a photoperiod of 12 hours quito-borne diseases by reducing man mos- – light and 12 hours dark in 80±10% relative quito contact. The most common insect re- humidity. The enriched wheat germ was used pellent is DEET (N, N-Diethyl-m-toluamide) as food source. Larvae of An. stephensi were moreover, there are several reports about of continuously available for the larvicidal and its toxicity against the skin, nervous and im- repellency experiments. Starved 7 to 10 days mune systems (Fradin et al. 1998, Katz et al. old females were used for the repellency 2008, Nerio et al. 2010). Insect repellents from tests and the early fourth-instars larvae used natural sources are a good and safe approach for the larval bioassays. for personal protection against the mosquito bites (Fradin et al. 1998). The repellent effect Plant materials of the plant essential oil has been examined The fresh branch and root of C. erecta col- against mosquito species in Iran (Oshaghi et lected in August 2011 from rural areas located al. 2003, Yaghoobi-Ershadi et al. 2006, Vatan- in western part of Ilam Province, Iran (33º doost and Hanafi-Bojd. 2008, Tavassoli et al. 1), and 2011). 46’ N, 46º 11’ E, elevation 1195) (Fig. was identified and authenticated by the De- Cionura erecta is classified in the family partment of Pharmacognosy, Faculty of Phar- , subfamily , tribe macy, Tehran University of Medical Sciences. Marsdenieae. This plant is a woody-based 50– 100cm high and up to 2m wide perennial with Essential oil isolation numerous herbaceous rambling stems, often Fresh roots (500g) of C. erecta were sub- twining at the tips, with copious poisonous jected to hydrodistillation using a modified milky juice and is widespread in the Medi- Clevenger-type (Pyrexfan®) apparatus for 3 terranean region (Tutin et al. 1976). This plant hour, the oil obtained was separated from known as poisonous composition which tradi- water and dried over anhydrous Na SO and tionally used for killing pest animals, there- 2 4 transferred into airtight vials at 5 °C. fore we used of it. Previous studies indicated that safranal, Analysis of essential oils (Z)-3-hexenyl benzoate, heneicosane were ob- Chemical composition of C. erecta was an- tained as major components from the essential alyzed using an Agilent 7890 5975 gas chro- oil of C. erecta in Turkey (Myrianthopoulos – matography mass spectrometer. With a HP- et al. 2007). - 5MS (5% Phenyl Methyl Silox) capillary col- This study was aimed at assessing the po- umn (30m×0.25mm, film thickness 0.25 , tential of plant essential oil and methanolic μm) split ratio, 1: 1, and using a flame ionization de- extract for possible use as larvicide or repel- tector. The GC was programmed at 50 °C for lent against An. stephensi under laboratory J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

2 min and then increased at 5 °C/min to 280 °C, repeat. The procedure for determination of ef- and finally held with an isothermal for 3min. fective dosages of the repellents was adopted The injector temperature was 280 °C. The flow by the standard method of American Society rate of the carrier gas was 1ml/min. The iden- for Testing and Material (ASTM 2000). tification of compounds was performed by The testing kit was made of plexiglas cube comparing their retention times and mass at dimension of 4×5×18cm having five circles spectra with mass spectra from Wiley library. with 29mm diameters. Before starting the test Additional identification was achieved by for determination of effective dosage, the ab- comparing linear retention indices, relative domen skins of rabbits were cleaned with al- to n-alkanes, to those from literature (Adams cohol and the kit was fixed on the abdomen. 2001). Details on the identification of volatile The criterion for selection of rabbits for re- compounds were reported in previous paper pellency tests was 10 landings or probes with- (Myrianthopoulos et al. 2007). in 30 seconds. Each of 5 adjacent cells of kit was provided with 5 female 7–10 days mos- Methanol extract of plant quitoes that randomly selected from a cage The branch and root of C. erecta were air- containing 150 starved mosquitoes. Five cir- dried at room temperature and 100g of plant cles were drawn on the rabbit's skin. The drawn were submitted to percolation separately with circles on the rabbit's skin were treated with Methanol (80%) during 3 days, and this pro- 25µl of essential oil diluted with absolute cedure were repeated for three times succes- alcohol at 2, 4, 8, 16, 32ppm with 4 repeat. sive and totally last nine day at laboratory tem- The serial dilutions were applied on 4 holes perature (22 to 25 °C). The extracts were next as well as the absolute ethanol was applied evaporated in a rotary evaporator (Heidolph in remaining control circle. The treated circles Persia®). were allowed to dry, and then test apparatus containing starved mosquitoes were fixed on Bioassays and larval mortality the treated skin. The counts of probing and The essential oil and methanol extract first biting were recorded at 1 minute intervals up dissolved in ethanol (99.0%) and methanol to 5 minutes. After each test, the mosquitoes (99.0%) respectively. The 400ml glass beak- were transferred to netted cups and the mor- ers were used for the treatment or untreated tality of mosquitoes was recorded after mainte- experiments. The early fourth instar larvae nance for 24 hours .The ED50 and ED90 val- were exposed to 10, 20, 40, 80 and 160ppm ues and regression parameters were analyzed and 40, 80, 160, 320, 640, 1280ppm of both using probit 79 program and the regression essential oil and methanol extract respectively lines were plotted in Microsoft Excel 2007. according to standard WHO procedure (WHO 1981). Ethical approval Animal experiments were performed after Repellency tests obtaining Institutional Animal Ethical Com- The white rabbits (Oryctolagus cuniculus) mittee’s approval from Tehran University of (laboratory reared albino females/ sex month) Medical Sciences. were used to determine both protection time and effective dosage. The 25%, 50% and 100% Results of essential oil from C. erecta was prepared using absolute ethanol as well as this solvent GC-mass analysis used for untreated group against An. stephensi The hydrodistillation of the C. erecta root on the shaved back of female rabbits with 4 gave oil in 0.16% (w/w) yield on fresh weight J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

material. A total of 19 compounds was 36.4% Effective doses in roots of C. erecta were identified (Table 1, The ED50 and ED90 values of C. erecta Fig. 2). The major components in root oil were essential oil were 10.12 and 23.01ppm with cedren-9-one (7.89%), alpha cadinol (5.67%), confidence interval ranged, 7.89–13.9 and eugenol (4.02%) and alpha muurolene (3.58%) 16.12–50.37 respectively (Table 4). respectively. Protection time Mosquito larvicidal activity The 25%, 50% and 100% essential oil C. The larvicidal activities of both essential erecta against An. stephensi on animal sub- oil and methanol extract of the C. erecta root ject were provided 2.0–3.15 hours protection. against An. stephensi larvae under laboratory The repellent failure time was ranged 2.5– conditions are shown in Table 2. Among the 4.25 hours (Table 3). five concentrations tested, the dosages of 320 ppm and 1280ppm of essential oil and meth- anol 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 methanol ex- tract against the larvae. The LC50 and LC90 values of C. erecta essential oil were 77.30 and 199.58ppm, and for methanol extract were recorded 250.38 and 490.00ppm, respectively. Fig. 1. The plant C. erecta (original)

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 .332 1391 5 Trans caryophyllon .21 1417 6 Trans caryophyllene .71 1473 7 alpha muurolene 3.58 1483 8 delta cadinene 1.52 1404 9 caryophyllene oxide 1.38 1540 10 Viridiflorol .55 1546 11 Silphiperfolenone .85 1551 12 Trans cadinene .64 1569 13 alpha cadinol 5.67 1577 14 Eudesmol 1.78 1584 15 Gama epoxy elemen .69 1598 16 Cedren-9-one 7.89 1633 17 isolongifolene-5-one 2.15 1644 18 Tetradecanol 1.34 1648 19 Cadalene .58 1677 36.44 *RI: Retention indices determined on HP-5 column J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

Fig. 2. GC-MS chromatogram of essential oil of C. erecta

Table 2. LC50 and LC90 values of different concentrations from essential oil and methanol extract of Cionura erecta roots against larvae of An. stephensi

Type of LC (ppm) LC (ppm) 2 2 a b ± SE 50 90 χ χ p-Value extraction ± 95%C.L. ± 95%C.L. (heterogeneity) table (df) 69.28 169.71 Essential -5.87 10.11 ± 0.34 77.30 199.58 5.419 * 11.345 (3) 0.01 oil 86.40 244.91 229.078 434.03 Methanol 4.40 10.54 ± 0.34 250.38 490.00 11.999 * 13.277 (4) 0.01 extract 273.66 571.00

* 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

ED ( mg/cm2) ED ( mg/cm2) 2 2 a b ± SE 50 90 χ χ p-Value ± 95%C.L. ± 95%C.L. (heterogeneity) table (df) 7.89 16.12 -3.59 3.575 ± 0.784 0.01012 0.02301 3.048* 13.277 (3) 0.01 13.89 50.37

* No heterogeneity J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

Discussion

Application of Larvicides and repellents are The repellency effect of the C. erecta es- generally accepted as the playing a significant sential oil against An. stephensi was first eval- role in preventing mosquito-borne diseases. uated under laboratory conditions. The mean In this study, major constituents of root protection time of 50% essential oil of C. erecta essential oil of C. erecta were evaluated. provided 2.15 hours protection against An. Cedren-9-one (7.89%), alpha cadinol (5.67%), stephensi. The figures for for ED50 and ED90 eugenol (4.02%) and alpha muurolene (3.58%) values were 10.12 and 23.01ppm respectively. were found as main compounds. The chem- The repellency effect of essential oils of ical ingredients of C. erecta essential oil was many plants has been evaluated against An. reported comprised 72 components, from stephensi (Ansari et al. 2000, Prajapati et al. which the main one considered as safranal 2005, Klun et al. 2006, Rajkumar and Jebanesan (16.8%), (Z)-3-hexenyl benzoate (6.1%), 2007, Mullai et al. 2008, Pandey et al. 2009, heneicosane (5.7%) linalool (4.8%) and Govindarajan et al. 2011, Prabhu et al. 2011). tricosane (4.4%) (Myrianthopoulos et al. 2011) The repellency effect of essential oils of both Some constitute was notfound in our study. Myrtus communis and Calendula officinalis According to the larvicidal assay, the es- had been reported and the ED50 values were sential oil and methanol extract of C. erecta 0.11 and 0.6 mg/cm2, respectively on human were effective against An. stephensi with subjects (Tavassoli et al. 2011). Other labor- LC50 and LC90 values of 77.30ppm and atory trial revealed the repellency of 3 chem- 250.38ppm, respectively. The bioassay of dif- ical and herbal repellents against An. stephensi. ferent herbal extracts has been stidied against The ED50 value of neem tree’s essential oil An. stephensi larvae in Iran. There is a report was 0.191mg/cm2 against field strain of mos- about the efficacy of the essential oil and quitoes (Vatandoost et al. 2008). methanolic extract of Eucalyptus camaldulensis The results indicated both the repellency against An. stephensi in which, the LC50 and of essential oil as well as the larvicidal effect LC90 values were found 89.85ppm and of C. erecta extract against An. stephensi. 397.75ppm, respectively (Sedaghat et al. 2010). The larvicidal activity of Azadirachta indica Acknowledgments extract against An. stephensi were gained 0.35 ppm and 1.81ppm respectively for LC50 and This work was supported by a grant (90- LC90 values (Vatandoost and Vaziri 2004). 04-27-16617) from Tehran University of Med- Also the LC50 and LC90 of Cupressus arizonica ical Sciences, Tehran, Iran. We are thankful to essential oil have been reported respectively the staff of Insectary of Culicidae, Department 79.30ppm and 238.89ppm against An. stephensi of Medical Entomology and Vector Control, (Sedaghat et al. 2011a). The larvicidal activ- School of Public Health, Tehran University ity of three plant from family Apiaceae have of Medical Sciences for the mass production been studied and the LC50 values of three of the mosquitoes for this study. The authors essential oils ranged from 20.10 to 120.95ppm declare that there is no conflict of interests. (Sedaghat et al. 2011b). In the other study, the efficacy of Kelussia odoratissima essen- tial oil was evaluated at dose of 10 ppm in- References duced 100% larval mortality, against larvae of both An. stephensi and Cx. pipiens Abbott WS (1925) A method of computing (Vatandoost et al. 2012). the effectiveness of an insecticide. J Am J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

Mosq Control Assoc. 3: 302–303. stephensi (Diptera: Culicidae). Asian Adams RP (2001) Identification of essential Pac J Trop Biomed. 1: 43–48. oil components by gas chromatog- Hadjiakhoondi A, Vatandoost H, Abousaber raphy/ mass spectroscopy. Allured, M, Khanavi M, Abdi L (2000) Chemi- Carol Stream IL, USA. cal composition of the essential oil of Alloway B, Ayres DC (1997) Chemical prin- Tagetes minuta L. and its effects on ciples of environmental pollution. CRC. Anopheles stephensi larvae in Iran. J Ansari MA, Vasudevan P, Tandon M, Razdan Med Plants. 7: 33–100. RK (2000) Larvicidal and mosquito re- Hadjiakhoondi A, Vatandoost H, Jamshidi A, pellent action of peppermint (Mentha Bagherj Amiri E (2003) Chemical con- piperita) oil. Bioresour Technol. 71: stituents and efficacy of Cymbopogon 267–271. olivieri (Boiss) bar essential oil against ASTM E (2000) Standard test methods for malaria vector, Anopheles stephensi. laboratory testing of non-commercial Daru. 11: 125–128. mosquito repellent formulation on the Hadjiakhoondi A, Vatandoost H, Khanavi M, skin. American Society for Testing and Abai MR (2005) Biochemical investiga- Materials, ASTM-E951-94 (Reapproved tion of different extracts and larvicidal 2000). activity of Tagetes minuta L on Anoph- Coluzzi M (1992) Malaria vector analysis and eles stephensi larvae. Iran J Pharm Sci. control. Parasitol Today. 8: 113–118. 1: 81–84. Davari B, Vatandoost H, Ladonni H, Shaeghi Hanafi-Bojd AA, Vatandoost H, Oshaghi MA, M, Oshaghi MA, Basseri HR, Enayati Haghdoost AA, Shahi M, Sedaghat MM, AA, Rassi Y, Abai MR, Hanafi-Bojd Abedi F, Yeryan M, Pakari A (2012) AA, Akbarzadeh K (2006) Comparative Entomological and epidemiological at- efficacy of different imagicides against tributes for malaria transmission and im- different dtrains of Anopheles stephensi plementation of vector control in south- in the malarious areas of Iran, 2004– ern Iran. Acta Trop. 121: 85–92. 2005. Pak J Biol Sci. 9: 885–892. Isman MB (2000) Plant essential oils for pest Davari B, Vatandoost H, Oshaghi MA, La- and disease management. Crop Prot. donni H, Enayati AA, Shaeghi M, Ba- 19: 603–608. sseri HR, Rassi Y, Hanafi-Bojd AA Isman MB (2006) Botanical insecticides, de- (2007) Selection of Anopheles stephensi terrents, and repellents in modern agri- with DDT and dieldrin and cross-re- culture and an increasingly regulated sistance spectrum to pyrethroids and world. Annu Rev Entomol. 51: 45–66. fipronil. Pest Biochem Physiol. 89: 97– Katz TM, Miller JH, Hebert AA (2008) Insect 103. repellents: historical perspectives and Finney DJ (1947) Probit analysis, a statistical new developments. J Am Acad Der- treatment of the sigmoid response curve. matol. 58: 865–871. Fradin MS (1998) Mosquitoes and mosquito Khanavi M, Vatandoost H, Khosravi-Dehaghi repellents. Ann of Internal Med. 128: N, Sanei-Dehkordi A, Sedaghat MM, 931–940. Hadjiakhoondi A, Hadjiakhoondi F Govindarajan M, Mathivanan T, Elumalai K, (2013) Larvicidal activities of some Krishnappa K, Anandan A (2011) Ovi- Iranian native plants against the main cidal and repellent activities of botan- malaria vector, Anopheles stephensi. ical extracts against Culex quinque- Acta Med Iran. 51: 141–147. fasciatus, Aedes aegypti and Anopheles J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

Klun JA, Khrimian A, Debboun M (2006) Re- Prabhu K, Murugan K, Nareshkumar A, pellent and deterrent effects of SS220, Ramasubramanian N, Bragadeeswaran Picaridin, and Deet suppress human S (2011) Larvicidal and repellent po- blood feeding by Aedes aegypti, Anoph- tential of Moringa oleifera against ma- eles stephensi, and Phlebotomus papa- larial vector, Anopheles stephensi Lis- tasi. J Med Entomol. 43: 34–39. ton (Insecta: Diptera: Culicidae). Asian Kunz SE, Kemp DH (1994) Insecticides and Pac J Trop Biomed. 1: 124–129. acaricides: resistance and environmen- Prajapati V, Tripathi AK, Aggarwal KK, tal impact. Rev Sci Tech. 13: 1249. Khanuja SPS (2005) Insecticidal, re- Lehane MJ (1991) Biology of blood-sucking pellent and oviposition-deterrent activ- insects. London, UK: Harper Collins ity of selected essential oils against Academic. Anopheles stephensi, Aedes aegypti and Mellanby K (1967) Pesticides and pollution. Culex quinquefasciatus. Bioresour Tech- Pesticides and pollution. nol. 96: 1749–1757. Mullai K, Jebanesan A, Pushpanathan T (2008) Rajkumar S, Jebanesan A (2007) Repellent Mosquitocidal and repellent activity of activity of selected plant essential oils the leaf extract of Citrullus vulgaris (cu- against the malarial fever mosquito curbitaceae) against the malarial vec- Anopheles stephensi. Trop Biomed. 24: tor, Anopheles stephensi liston (diptera 71–75. culicidae). Eur Rev Med Pharmacol Sci. Sedaghat MM, Sanei-Dehkordi A, Abaie MR, 12: 1–7. Khanavi M, Mohtarami F, Saim abadi Myrianthopoulos V, Fokialakis N, Melliou E, Y, Vatandoost H (2011b) Larvicidal Mitaku S (2007) Chemical Composi- Activity of Essential Oils of Apiaceae tion Of The Essential Oil Of Cionura Plants against Malaria Vector, Anoph- Erecta (Asclepiadaceae) Inforescences. eles stephensi. Iran J Arthropod-Borne J Essent Oil Res. 19: 266–268. Dis. 5(2): 51–59. Nerio LS, Olivero-Verbel J, Stashenko E Sedaghat MM, Sanei-Dehkordi A, Khanavi M, (2010) Repellent activity of essential Abai MR, Hadjiakhoondi A, Mohtarami oils: a review. Bioresour Technol. 101: F, Vatandoost H (2010) Phytochemistry 372–378. and larvicidal activity of Eucalyptus Oshaghi MA, Ghalandari R, Vatandoost H, camaldulensis against malaria vector, Shaeghi M, Kmali-Nejad M, Tourabi- Anopheles stephensi. Asian Pac J Trop Khaledi H, Abolhassani M, Hashem- med. 3: 841–845. zadeh M (2003) Repellent effect of ex- Sedaghat MM, Sanei-Dehkordi A, Khanavi tracts and essential oil of Citrus limon M, Abai MR, Mohtarami F, Vatan- (Rutaceae) and Melissa officinalis doost H (2011a) Chemical composi- (Labiatae) against main malaria vector, tion and larvicidal activity of essential Anopheles stephensi (Diptera: Culicidae) oil of Cupressus arizonica E.L. Greene in Iran. Iran J Public Health. 32: 47–52. against malaria vector Anopheles ste- Pandey SK, Upadhyay S, Tripathi AK (2009) phensi Liston (Diptera: Culicidae). Phar- Insecticidal and repellent activities of macognosy Res. 2: 135–139. thymol from the essential oil of Trachy- Tavassoli M, Shayeghi M, Vatandoost H, spermum ammi (Linn) Sprague seeds Khoobdel M, Salari M, Ghaderi A, Rafi against Anopheles stephensi. Parasitol F (2011) Repellency Effects of Essen- Res. 105: 507–512. tial Oils of Myrtle (Myrtus communis), Marigold (Calendula officinalis) Com- J Arthropod-Borne Dis, E Mozaffari et al.: Chemical Composition …

pared with DEET against Anopheles and Culex pipiens (Diptera: Culicidae). stephensi on Human Volunteers. Iran J Exp Parasitol. 132: 470–474. Arthropod-Borne Dis. 5: 10–22. Vatandoost H, Vaziri VM (2004) Larvicidal Tutin TG, Heywood VH, Burges NA, Valen- activity of a neem tree extract (Neemarin) tine DH (1976) Flora europaea. Cam- against mosquito larvae in the Islamic bridge University Press. Republic of Iran. East Mediterr Health Vatandoost H, Hanafi-Bojd AA (2005) Current J. 10: 573–581. resistant status of Anopheles stephensi Vatandoost H, Zahirnia AH (2010) Respon- Liston to different larvicides in Hor- siveness of Anopheles maculipennis to mozgan Province, southeastern Iran. different imagicides during resurgent Pak J Biol Sci. 8: 1568–1570. malaria. Asian Pacific J Trop Med. 3: Vatandoost H, Hanafi-Bojd AA (2008) La- 360–363. boratory evaluation of 3 repellents WHO (1981) Instruction for determining the against Anopheles stephensi in the Is- susceptibility or resistance of mosquito lamic Republic of Iran. East Mediterr larvae to insecticides. World Health Health J. 14: 260–267. Organization-VBC. Vatandoost H, Mashayekhi M, Abai MR, WHO (1985) Resistance of vectors of dis- Aflatoonian MR, Hanafi-Bojd AA, ease to pesticides. 10th report of the ex- Sharifi I (2005) Monitoring of insecti- pert committee on vector Control. cides resistance in main malaria vectors WHO (2008) World Malaria Report. World in a malarious area of Kahnooj district, Health Organization. Kerman Province, southeastern Iran. J Yaghoobi-Ershadi MR, Akhavan AA, Jahani- Vector Borne Dis. 42: 100–108. fard E, Vatandoost H, Amin GH, Vatandoost H, Sanei Dehkordi A, Sadeghi Moosavi L, Ramazani ARZ, Abdoli H, SMT, Davari B, Karimian F, Abai MR, Arandian MH (2006) Repellency ef- Sedaghat MM (2012) Identification of fect of Myrtle essential oil and DEET chemical constituents and Larvicidal against Phlebotomus papatasi, under activity of Kelussia odoratissima Labo-ratory Conditions. Iran J Public Mozaffarian essential oil against two Health. 35: 7–13. mosquito vectors Anopheles stephensi