Bioresource Technology 98 (2007) Short communication

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1 Bioresoure Tehnology 98 (7) Short ommunition The use of Eulyptus teretiornis Sm. (Myrtee) oil (lef extrt) s nturl lrviidl gent ginst the mlri vetor Anopheles stephensi Liston (Dipter: Culiide) Sengottyn Senthil Nthn * Post Grdute nd Reserh Deprtment of Biotehnology, Viveknndh College (W), Trihengode, Nmkkl, Tmil Ndu 637 5, Indi Plnt Environment Division, Honm Agriulturl Reserh Institute (HARI), Ntionl Institute of Crop Siene (NICS), Rurl Development Administrtion (RDA), #381 Songhk-dong, Iksn, Chonuk 5-, Repuli of Kore Reeived 1 April 6; reeived in revised form 3 July 6; epted 4 July 6 Aville online 25 Septemer 6 Astrt Seondry metolites otined from the indigenous plnts with proven mosquito ontrol potentil n e used s n lterntive to syntheti insetiides under the integrted vetor ontrol. The essentil oil extrt from the forest redgum, Eulyptus teretiornis Sm. (Myrtee) ws tested ginst mture nd immture mosquito vetor Anopheles stephensi Liston (Dipter) under lortory ondition. The extrt showed strong lrviidl, pupiidl nd dultiidl tivity. The lef oil extrts showed high iotivity t high doses. Results otined from the lortory experiment showed tht the lef extrts suppressed the pupl nd dult tivity of Anopheles stephensi t higher doses. In generl, first nd seond instr lrve were more suseptile to ll tretments. Cler dose response reltionships were estlished with the highest dose of 1 ppm plnt extrt evoking lmost 1% mortlity. The results otined suggest tht, in ddition to their mediinl tivities, E. teretiornis n lso serve s nturl mosquitoide. Ó 6 Elsevier Ltd. All rights reserved. Keywords: Mosquito vetor; Forest redgum; Biology, mortlity, lrviide, dultiide; Oviposition deterrene 1. Introdution Mosquitoes re vetors of etiologi gents of mlri, filrisis, nd virl disese. Anopheles stephensi Liston (Dipter) is the primry vetor of mlri in Indi nd other West Asin ountries, nd improved methods of ontrol re urgently needed (Gilles nd Wrrell, 1993; Collins nd Pskewitz, 1995; Burfield nd Reekie, 5). Reently, otnil nd miroil insetiides hve een inresingly used for mosquito ontrol euse of their effiy nd doumented non-toxi effets on non-trget orgnisms * Address: Plnt Environment Division, Honm Agriulturl Reserh Institute (HARI), Ntionl Institute of Crop Siene (NICS), Rurl Development Administrtion (RDA), #381 Songhk-dong, Iksn, Chonuk 5-, Repuli of Kore. Tel.: ; fx: E-mil ddresses: senthilklidr@hotmil.om, senthil@rd.go.kr (Asher et l., 1995; Amlrj et l., ; Gunsekrn et l., 4; Senthil Nthn, 6). The development of Inset Growth Regultors (IGR) hs drwn muh ttention s seletive ontrol of insets of griulture, medil nd veterinry importne nd hs produed mortlity due to their high neurotoxi effets (Al-Shrook et l., 1991; Senthil Nthn et l., 5, 6,). Reent studies hve lso stimulted the investigtion of insetiidl properties of hemils derived from plnt mteril nd onluded tht they re environmentlly sfe, degrdle, nd trget speifi. Indeed, mny mediinlly importnt plnt extrts were studied for their effiy to kill lrve of different speies of mosquito (Sxen nd Sumithr, 1985; Kumr nd Dutt, 1987; Chrindy et l., 1999; Mrkouk et l., ; Tre et l., 4) nd other medilly importnt prsites (Adel-Shfy nd Zyed, 2; Borges et l., 3; ICMR, 3) /$ - see front mtter Ó 6 Elsevier Ltd. All rights reserved. doi:1.116/j.iorteh

2 S. Senthil Nthn / Bioresoure Tehnology 98 (7) The otnil fmily Myrtee is potentil soure for mosquito repellent ompounds. Leves of Eulyptus teretiornis Sm. re rih in the ompound ineol, (Frnih, 1985). E. teretiornis hs long een reognized for its insetiidl properties; espeilly its mosquito repellent tivity (Wtne et l., 1993; Coret et l., 1995; Troulsi et l., 5) ut hs yet to e extensively nlyzed. Extrts from the Eulyptus leves hve shown exellent lrviidl nd repellent properties ginst mosquito vetors while t the sme time eing very eo-friendly (Wtne et l., 1993). My present investigtion demonstrtes the effiy of extrts of E. teretiornis in killing lrvl nd dult stges of A. stephensi under lortory onditions. 2. Methods 2.1. Mosquito ulture A. stephensi eggs were olleted round the Viveknndh College Cmpus, Nmkkl Distrit, Tmil Ndu nd rered in plsti nd enmel trys in tp wter. They were mintined in growth hmer nd ll the experiments were rried out t 27 ± 2 C, 75 85% reltive humidity with 14:1 light nd drk photoperiod. Lrve were fed diet of Brewers yest, dog isuits nd lge olleted from ponds in the rtio of 3 (Brewers yest): 1 (dog isuits): 1 (lge). Pupe were trnsferred from the trys to up ontining tp wter nd pled in sreened ges ( m) where dults emerged. Adults of A. stephensi were rered in m glss ges. Adults were ontinuously provided with 1% surose solution in jr with otton wik. On dy five post-emergene, dult femles were deprived of sugr for 12 h then provided with mouse pled in resting ges overnight for lood feeding. Adult mosquitoes were mintined t the sme environmentl ondition s lrve Plnt extrts E. teretiornis leves (mture) were olleted from five trees pproximtely out months old in nturl forests of Kolli hills, Nmkkl Distrit, Tmil Ndu, Indi. The leves were piked out during the morning. The essentil oils were isolted y stem-distilltion using Clvenger pprtus, dried over nhydrous sodium sulphte, nd stored in mer-olored vils t 5 C until required for further work Biossys nd lrvl mortlity First to fourth instr lrve nd pup were exposed to test onentrtions of 1,,, nd 1 ppm of the oil in distilled wter for 24 h ording to stndrd WHO proedure (1981). As the essentil oil does not dissolve in wter it ws first dissolved in ethnol (99.%). The test medium (2 ml glss eker) ws prepred y dding 1 ml of pproprite dilution of essentil oil in ethnol nd mixed with 249 ml of wter to mke up 2 ml of test solution (Dhrmgdd et l., 5). The lrve were fed dry yest powder on the wter surfe ( mg/l). Ethnol served s ontrol (1%). A minimum of lrve/onentrtion ws used for ll the experiments. The ded lrve were ounted fter every hour, nd perentge mortlity is reported from the verge for the five replites tken. The lethl onentrtions (LC nd LC ) were lulted using Proit nlysis (Finney, 1971). The perentge mortlity ws lulted y using the formul (1) nd orretions for mortlity when neessry were done y using Aot s (1925) formul. Perentge of mortlity Numer of ded lrve ¼ Numer of lrve introdued 1 ð1þ 2.4. Adultiidl ssy A. stephensi dults (1 individuls) were exposed to filter pper ( mm, Advnte Toyo, Jpn) treted with onentrtions of 1,,, nd 1 ppm E. teretiornis oil extrt. The pper ws kept inside the eker. Control insets were exposed only to ethnol treted pper nd muslin loth. A mortlity ount ws tken fter 24 h. The experiment ws repeted five times Oviposition ssy During the tests, the groups of femles (1 individuls fed on mie lood) were kept seprte for 48 h in ge mesuring m. The ge ontined six glss jrs with 1,,, nd 1 ppm onentrtions of the E. teretiornis. Ethnol ws used s the ontrol (five replites). Ten grvid femles were given hoie etween treted nd ontrol jrs. After the eggs were ounted, the oviposition deterrene index (ODI) (Hwng et l., 1982) ws lulted y using the formul (2): Nt Ns ODI ¼ Nt þ Ns 1 ð2þ where, Nt = totl numer of egg rfts in test solution, Ns = totl numer of egg rfts in ontrol Sttistil nlysis The lethl onentrtion ws lulted y using Proit nlysis (Finney, 1971). Mortlity ws orreted y using Aot s (1925) formul. Dt from mortlity nd oviposition deterrene were nlyzed with ANOVA of rsine squre root trnsformed perentges. Differenes etween mens were onsidered signifint t P 6.5 (SAS Institute, 1). In the Tukey s test, desending order ws used. The highest different vlues from verge, deteted y sttistil testing were mrked with letter, the next text lower with nd ontinued ordingly (Snedeor nd Cohrn, 1989).

3 1858 S. Senthil Nthn / Bioresoure Tehnology 98 (7) Conentrtion in ppm Results LC LC I instr II instr III instr IV instr Pup Adult Stges of An. stephensi Fig. 1. Lethl onentrtion (LC ) nd (LC )ofe. teretiornis ginst lrvl, pupl nd dult of A. stephensi (vlues re men of five replites with ±(SEM) stndrd error). Exposure of E. teretiornis extrt in mosquito lrvl diet redued lrvl survivility nd inresed mortlity in ll lrvl instrs. The LC nd LC vlues of E. teretiornis oil ginst the mlri vetor re shown in Fig. 1. First (18.3 nd 51.6 ppm for LC nd LC, respetively) nd seond instr (23.8 nd 63.9 ppm for LC nd LC, respetively) lrve were more suseptile with lest LC vlues (Fig. 1). The 1 ppm onentrtion of lef extrt oil killed more thn 95% of first instrs, (F = ; df = 4; Mortlity in perentge d P <.1), 94% of seond instrs (F = ; df = 4; P <.1), 92% of third instr (F = ; df = 4; P <.1), % of fourth instrs (F = ; df = 4; P <.1) nd 88% of pupe (F = ; df = 4; P <.1) (Figs. 2 nd 3). The sme trend ws lso oserved on perentge mortlity of dult of A. stephensi (Fig. 3). At higher onentrtions ( nd 1 ppm), the lrve showed irregulr movement for some time nd then died Control 1 ppm ppm ppm ppm 1 ppm IV P A IV P A IV P A IV P A IV P A IV P A E.teretiornis tretment onentrtion Fig. 3. Perentge of fourth instr, pupe nd dult mortlity of A. stephensi fter tretment with E. teretiornis. Mens (±(SEM) stndrd error) followed y the sme letters within sme mosquito stge of indite no signifint differene (P 6.5) in Tukey test (IV, P, nd A indite fourth instr, pupe nd dult respetively). Mortlity in perentge d d Control 1 ppm ppm ppm ppm 1 ppm I II III I II III I II III I II III I II III I II III E.teretiornis tretment onentrtion Fig. 2. Perentge of first to third instr lrvl mortlity of A. stephensi fter tretment with E. teretiornis. Mens (±(SEM) stndrd error) followed y the sme letters within sme instr of indite no signifint differene (P 6.5) in Tukey test (I, II, nd III indite first, seond nd third instr respetively). Oviposition deterrene index in perentge 1 Control 1 ppm ppm ppm ppm 1 ppm E.teretiornis tretment onentrtion Fig. 4. Ovipositionl deterrene index (ODI) of A. stephensi fter the tretment with E. teretiornis. Mens (±(SEM) stndrd error) followed y the sme letters within rs of indite of no signifint differene (P 6.5) in Tukey test.

4 S. Senthil Nthn / Bioresoure Tehnology 98 (7) t the ottom of the ekers. No pupl or dult emergene ws oserved mong the tretments s lmost 1% mortlity ourred within 24 h. A signifint differene ws oserved etween higher onentrtions ( nd 1 ppm) nd lower onentrtions ( nd ppm). The effet on lrvl, pupl nd dult mortlity ws onentrtion-dependent. There ws grdul derese in oviposition when femle A. stephensi were treted with E. teretiornis oil extrt in lower dose tretments ut fter tretment with more thn ppm, the effet ws more pronouned (Fig. 4). Mximum oviposition deterrene ws oserved in 1 ppm nd it ws signifintly different from other lower onentrtion (exept ppm) tretments (F = 24.95; df = 4; P <.1). 4. Disussion Results on the lrvl mortlity nd oviposition deterrene of the ppm extrt on the dult A. stephensi reported in the present study, onfirm their potentil for ontrol of the mosquito popultions. These properties n e exploited to protet the immedite environment of the user or the user from the ites of hrmful insets, whih my e vetors of disese. It ws lso oserved tht first nd seond instr lrve were more suseptile to the lef extrt (Fig. 2). Exposure of A. stephensi lrve to su-lethl doses of neem extrts in the lortory prolonged lrvl development, redued pupl weight, high oviposition deterrene nd high mortlity (Su nd Mull, 1999; Wndsheer et l., 4; Troulsi et l., 5). The diret nd indiret ontriution of suh effets to tretment effiy through redued lrvl feeding nd fitness need to e properly understood in order to improve the use of otnil insetiides for mngement of A. stephensi. These nd other nturlly ourring insetiides my ply more prominent role in mosquito ontrol progrms in the future (Amlrj et l., ; Gunsekrn et l., 4; Wndsheer et l., 4). After E. teretiornis tretment t higher dose, the lrve died immeditely efore the pupl stge. The lrve eome norml nd irregulr in movement. The lrviidl mode of tion of essentil oils ws investigted y Coret et l. (1995) who noted the suseptiility of mosquito lrve nd pupe to surfe mterils entering their trhel system, oserving tht essentil oils inresed the tendeny to trhel flooding nd hemil toxiity. However, Senthil Nthn et l. (5) onsidered pure limonoids of neem seed, testing for iologil, lrviidl, pupiidl, dultiidl, nd ntiovipositionl tivity, A. stephensi. Lrvl mortlity ws dose-dependent with the highest dose of 1 ppm zdirhtin evoking lmost 1% mortlity, ffeting pupiidl nd dultiidl tivity nd signifintly deresed feundity nd longevity of A. stephensi. The tretment ompletely inhiited the lrvl, pupl nd dult developments. The limonoids lso interfered with oviposition, egg hthility, nd exhiited growth inhiiting effet ginst lrve nd good mortlity effet ginst dults of A. stephensi. The sme results were lso otined when Meli zedrh L. lef nd seed extrts were used (Senthil Nthn et l., 6). Although the otnil insetiides re the lesser of mny hzrds in terms of generl pestiide toxiities, they re toxins nonetheless. All toxins used in pest ontrol pose some hzrds to the user nd lso to the quti environment (Kreutzweiser, 1997). Hene this reserh is minly foused on finding newer insetiides whih will e more effetive, iodegrdle nd lso esily ville t low ost. Aknowledgements The uthor would like to thnk Prof. John F. Anderson nd two nonymous reviewers for their omments on n erlier version of the mnusript Mr. Krthikeyn, Tehniin for his vlule support during the reserh period nd lso uthor s grdute students Deny K George, L. Sugny nd A. Nrmdh for their voluntry help during the projet reserh period. Finnil help to the uthor from the HARI, RDA, NICS to onlude this work is grtefully knowledged. Referenes Aot, W.S., A method of omputing the effetiveness of n insetiide. J. Eo. Entomol. 18, Adel-Shfy, S., Zyed, A.A., 2. 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