282 Jounrer,or r:npArr,rnRrceN Mosqurro CoNrRor, Assocrerron VoL.7, No.2

LARVICIDAL ACTIVITY OF MINUTA (MARIGOLD) TOWARD AEDES AEGYPTI

M. M. cREEN,l J. M. SINGER,' D. J. SUTHERLAND, nNo C. R. HIBBEN3

ABSTRACT. The steam distilled oils of 3 speciesof mari gold.,, T. erecta and T. minuta, were tested for larvicidal activity toward thirdin starAedes degwti;"activity at fb ppm was demonstrated only for T. minuta.. The larvicidal property of the whole oil"dispersedin water persisted for at least 9 days.The terpene,ocimenone, which is a part of the whole oil, was found to l" taruiciaai o"it;t;;igh"; concentration than the whole oil and to lose its activity *ithi.r z4 h after dispersrt i" rial... ffrr.. results-suggest.a potential utilization of oil of T. minuta or its componentsfor th'e control of Ae.aegypii and other speciesof mosquitoes.

INTRODUCTION MATERIALS AND METHODS In folklore claims(Neher 1968),marigolds are Seeds of Tagetespatula Linn. and Togetes commonly believedto be useful for insect erecta Linn. were obtained from W. Atlee Bur- control. The scientific literature givescredibility pee Co.; seedsof T. minuta were provided bv J. to thesebeliefs in at least 3 instances:a nema- O. Kokwaro, University of Nairobi, Kenya, and ticide has been isolated as a root exudate from by the Brooklyn Botanic GardenResearch Cen- various speciesof marigolds (Uhlenbroek and ter. The plants were grown in a soillessmix Bijloo 1958,1959; Downum and Towers 1988); (Redi-EarthPeat-Lite Mix, GraceHorticultural the oil of Tagetesminuta Linn. is reported to Products) in an automatically ventilated, contain componentsthat act as a juvenile hor- screenedresearch greenhouseunder natural monemimic (Saxenaand Srivastava1973): and Iight (6,458-16,146lumens/m') supplemented the oil of T. minuta is reported to act as a fly by 8 h additional illumination (I2,9t7-t6,t46 ovipositionrepellent (Monnig 1986).However, Iumens/m2,high pressuresodium lamps), 24"C oil of ?. rninuta is not a mosquito repellent day/16'C night temperature cycle, b0-85% RH. (McCullough and Waterhouse1942). Young plants were fertitized with a 15-30-15 In an investigation stimulated by folklore food and again at the beginning of flow- claims,Maradufu et al. (1978)reported that the ering. No pesticides were applied to the test steam distilled oil of T. minuta contained a plants. Additional marigolds were grown in a mosquito larvicide active against Aedes aegypti fenced field plot in 24-m rows of rototillei (Linn). The activeprinciple wasassigned to bE- Charlton loam soil (pH 6.1). Seedlingswere ocimenone,a long-knownnatural componentof thinned to 2-5 cm apart. The plants were fertil- marigold oils (Jones and Smith 1925, Jones ized with a 5-10-5plant food at 0.4b kg/9.8 m2 1926, Naves 1948). Unfortunately, bE-ocime- as a side dressingat the beginning of flowering. none isolated from the plant was not stable in Supplementarywatering was applied during "in- dry water.According to Maradufu et al. (1g28), periods. No pesticides or herbicides were ap- troduction of fresh larvae to a 24-h-old mixture plied. of 5E-ocimenone-waterdid not causethe death The above-groundparts and some roots from of the larvae." However, fresh larvae were not marigolds in the greenhouseand field plot were addedto a 24-h old mixture of the whole oil. In harvested during the period of maximum flow- our work, this important control experiment was ering. The plant material was placedin plastic carried out. The results of this and related ex- bags and stored for a few days at 30'C until perimentsshow that 5E-ocimenoneis not solelv treatment. responsiblefor the mosquito larvicidal activity The stems, Ieavesand flowers were triturated of the steamdistilled oil of ?. minuta and that with about 400 ml of distilled water to a medium- other stable componentsprobably are responsi- fine texture in a commercial stainless steel ble. blender (Scovill, Inc.). The mixture was then suction filtered through a Buchner funnel and Whatman ffl fiter paper. The drained, moist masswas transferred to clean,labeled aluminum I Department of Chemistry, Polytechnic University, foil freezer containers. The filtrate was reused 333Jay St., Brooklyn, NY 11201. ' throughout this procedure Mosquito Research& Control, Department of En- for each batch of tomology, Cook College, Rutgers University, New plants processedand replenishedto original vol- Brunswick,NJ 08903. ume as needed.Prior to sealingthe containers, 3 Brooklyn Botanic Garden ResearchCenter, ?12 this filtrate was apportioned to each container Kitchiwan Road,Ossining, NY 10562. of blended material; all pans were filled to weigh JUNE 1991 MARTcoLDToxrcrrv ro MoSQUIToES 283 approximately 2 kg. The sealedcontainers were dilution a final concentration of the specified storedat -20'C. ppm (v/v) of oil was achieved. The essentialoil was obtained in one stageof Aliquots of 224 ml of distilled water were the work, by classicalsteam distillation of 500 g addedto 250-mltest beakers.One ml of the test portions of chopped, frozen plant material for solution was added to each beaker, followed by 4-6 h. The distillate was extracted twice with 2Slawaein 25 ml of distilledwater. The beakers pentane, and the pentane solution stored under were arranged on a tray, coveredIoosely with a nitrogen in amber bottles, with Teflon cap- sheet of aluminum foil and placed in an incu- liners at -20'C until neededfor analysis.Alter- bator at 30'C. At intervalsthereafber, the beak- natively, 200-300 g of chopped, frozen plant ers were removed and the number of dead and material was steam distilled for 4 h, with pen- living individuals and their respectivestages of tane as the organic solvent,in a simultaneous development were recorded. Mortality was es- steam distillation apparatus (J&W Scientific tablished as the lack of head-to-tail flexion in was minimized responseto tapping the beakerwith the end of Corporationa).Loss of volatiles "Sluggishness" by use of a cold-finger containing a dry ice/ a pencil. was characterizedby methanolslurry, installedon top of a condenser. the Iarvae appearing at the surface, pointed The pentanecontaining oil wasstored in the 50- downward, without consistent head-to-tail flex- rnl conical flask used with the extractor and ion responseto beaker tapping; failure of slug- kept under nitrogen at -20'C until needed. gish larvae to respond was confirmed by prod- Prior to analysisthe oil was obtained by removal ding the larvae with a pencil tip. of the pentane on a rotary thin film evaporator without heat. All oils were stored in 0.3-ml RESULTS AND DISCUSSION ReactiVials(Pierce Chemical Co.) with Teflon- faced capliners under nitrogen at -20'C until A mixture of Z and E ocimenonestereoiso- analysis. mers (Weyerstahlet al. 1986)was synthesized Ocimenone, 2,6-dimethylocta-2,5,7-trien- - accordingto the Iiterature(deVilliers et al. 1971, one, was prepared as a mixture of isomers ac- Adams et al. 1975);these authors suggestthat cording to the literature (deVilliers et al. 1971, the isomersare isolatedin a ratio of 9:1. The Adams et al. 1975). isomer mixture we obtained was subjectedto gas Mosquito larvae from the Rockefeller strain chromatography/mass spectrometry (GC/MS), of Ae. aegypti, which has been in continuous and it was discoveredthat the major chromato- culture at Rutgers University since 1935, were graphic peak previously assignedto one of the usedin thesetests. Between 250-500 Iarvae were isomers is actually a mixture of 2 ocimenone hatched3 daysprior to the bioassayin enameled isomersin the approximateratio of 3:2 and that pans (33 x 2t.6 x 5 cm) containing 1 liter of the minor chromatographicpeak is not an oci- distilled water; approximately 6-8 pans were menone. The fragmentation patterns closely required for eachbioassay. The larvae developed match those reported by Hethelyi et al. (1987), at 27"C and were provided daily approximately deVillierset al. (1971)and Adams et al. (1975). 300 mg/pan/day of ground hog supplement (Ag- Extensive chromatographicexperiments were in way). The water was skimmed daily to remove agreement with these conclusions (Singer surface scum. The pans were transferred to an 19875). incubator at 30'C for the final 12 h prior to the The steam-distilledoil of ?. minuta was also test. At the time of the bioassaythe larvae were subjectedto GC/MS analysis,and the chromat- at mid-3rd instar. Larvae from severalpans were ographic peak of identical retention time to the washedin a cloth strainer with 1 liter of distilled major peak from the synthetic ocimenone was water and transferred to clean pans containing shown to contain an identical isomeric mixture. 1 liter of distilled water. It is unlikely that the identity ofthe ratio ofthe The test substanceswere volumetrically di- mixtures arises from an identical production of luted in absoluteethanol (USP grade) at a stock isomeric ocimenonesin the plant and in the in concentrationof 2.5 pI/ml (v/v) suchthat upon ultro synthesis, but rather that isomerization (Boehmeet al. 1963)has led both systemsto the samepoint of equilibrium. For the bioassaywork the ocimenonewas usedas this mixture of iso- a The activity of T. patuln oil in the bioassays was strongly dependent on the method of steam distilla- tion. Classical steam distillation led, irreproduceably, to high larvicidal activities compared with the simul- taneous apparatus. This difference could be due to the 5 Singer, J. M. 1987. Mosquito larvicidal investiga- decreased potential of carryover of the still pot con- tion of oil of marigold. Doctoral Thesis, Pol5rtechnic tents into the distillate in the simultaneous svstem. University, Brooklyn, NY. JounNer,oF THEAlrnnrceu Moseutro ConrRor,AssocrarroN VoL.7, No.2

Table 1. Larvicidal activities of steam distilled Tagetesoils and control substances to Aedesaegypti.

Vo mortality + SD at indicated contact time Concentration Substance (pp-) 24h 48h 72h 96h T. minuta oil 10 69+26 96+9 99-f5 99-f3 20,24 88. 88 92,96 100, 96 0.4 No mortality observedd T. patula oil 10 J=J I+2 / =,J 11 tt) T. erecta oiI 10 No mortality observedd CrHoOH" No mortality observedd Ocimenonesb 10 18+3 68+23 77+24 78+22 40 96+5 100 100 100 " 1 ml of CTHsOH to 224 ml in test beaker. b Synthetic mixture of isomers. See text. " Two replicates. d In various replicate observations the standard deviation could not distinguish mortalities differing from zeto,

Table 2. Time stability of the larvicidal activity of Tagetes minuta whole oil and ocimenones against Aedes aegypti.

7o mortality + SDb at indicated contact time Concentration Substance (ppm., Time (h)' 24h 48h 72h Ocimenones 40 qA 0 7+-71 19-F1 10 0 28 )- 34 36 +- 28 T. minuta oil 10 24 100 100' 100' 10 72 95{-5 100 10 216 98+1 100 C,HsOH 72 0 16+8 216 0 10+2 " Time betweenpreparation of test solution and addition of larvae. b Time after addition of larvae to test solution. " Removeddead larvae and added 25 fresh larvae in 25 ml of distilled water. d 1 ml of CzHsOHto 224 ml of water.

Table 1 shows the larvicidal activity for the supportthe claim of Maradufu et al. (1978)that oils of 3 speciesof marigolds,an ethanol control ocimenoneis the soleactive principle in the oil. and the ocimenoneisomers described above, as One of the key findings of Maradufu et al. a function of time. These results generally agree (1978)was the lossof activity of the ocimenone with the previous findings of Maradufu et al. isolated from the plant in the aqueoustest mix- (1978) who tested only the oil of T. minuta. tures with time. If found for the whole oil, this From these data, T. minuta is highly larvicidal instability would preclude usageof marigold oil compared with the other species T. patula, T. or its derivativesfor controlling mosquito larvae. erecta and with the control. We observed that To reexaminethis aspect,we mixed the appro- the larvae were sluggishbefore death. This slug- priate concentrationsof T. minuta whole oil or gish behavior affected proportionately larger ocimenonewith water, incubated the mixture at percentagesoflarvae at one ofthe lower concen- 30'C for variousperiods of time, and addedthe trations used (4 ppm). However,by 72 h both Ae. aegypti larvae. Table 2 indicates that oci- concentrationlevels (4 and 10 ppm) produced menone, as reported by Maradufu et al. (1978), about the same mortality. At the 0.4 ppm level in aqueous suspension for 24 h substantially of T. nr.inutaoil there was essentially no mor- losesits larvicidal activity (comparewith Table tality; however, many failed to pupate and 1) at 10 ppm and 40 ppm. In contrast, in our emergeas adults in contrast to ethanol controls. studies the whole oil maintains its activity in Results indicated that the oil of ?. minuta acts mixtures of whole oil and water for as long as 9 as a larvicide at a lower concentration than does days (216 h). Parallel chromatographicexperi- the synthetic ocimenone;only at 40 ppm did ments (Singer 19875)on both the whole oil and ocimenone produce high initial mortality. The the synthetic ocimenone in aqueous mixtures greater toxicity of the ?. minuta oil does not indicate that the ocimenone is almost entirely JUNE 1991 MlRrcor-o Toxlclrv ro MoSQUIToES 285

decomposed after 24 h in both the synthetic REFERENCES CITED samplesand in the oil. We have shown that synthetic ocimenoneis P. Bhatnager, R' C. Cookson and R. larvae than the plant Adams, D. R., S. Iesstoxic to the mosquito M. Tuddenham. 19?5. Simple synthesis of the atlan- whole oil and that the plant whole oil on stand- tones, the ocimenones, the tagenones, and filifolone ing in water maintains its larvicidal activity long from isoprene. J. Chem. Soc. Perkin I.1975:.1741- after ocimenoneloses its activity. Therefore, we r743. concludethat the larvicidal activity of the steam Bailey,L. H. andE. Z. Bailey.1976. Hortus third. A distilled oil of ?. minuta against Ae. aegypti compiete dictionary of plants cultivated in the Canada, Revised. Macmillan, be attributed only to ocimenone(Mar- United States and cannot New York. adufu et al. 1978). Boehme, E. E., V. Thaler and M. C. Whiting. 1963. The results in Table 2, demonstrating in a Synthetical studies on terpenoids. Part II. The Iaboratory environment that the whole oil in structure oftagetone. J. Chem. Soc. 1963:2535-2540. water retains Iarvicidal activity for at least 9 Brattsen, L. B., C. W. Holyoke, J. R. Leeper and K. days, suggest that field evaluations are war- F. Raffa. 1986. Insecticide resistance: challenge to pest and basic research. Science. ranted. In preliminary plastic barrel tests (48 management ppm of 231:1255-i260. liter) at Rutgers University using 40 Casida, J. E. 19?6. Prospects for new types of insecti- whole oil, Culexpipiens Linn. was controlled. It cides. pp. 349-366. In: R. L. Metcalf and J. J- is likely that the material active both against McKelvey (eds.). The future for insecticides: needs Ae. aegypti and Culex would also be active and prospects. Wiley, New York. against Anophelesmosquitoes. This likelihood, Clements, A. N. 1963. The physiology of mosquitoes. in with the ease of obtaining the Macmillan, New York. combination J. L. 1976. Insects and history. geographic Cloudsley-Thompson, active oil with steam and the wide St. Martins Press, New York. rangeof this commonplant (Neher 1968,Cloud- Dame. D. A.. R. E. Lowe, G. J. Wichterman, A. L. sley-Thompson1976, Bailey and Bailey 1976, Cameron, K. R. Baldwin and T. W. Milier. 1976. Gray 1887),suggests that this plant could play Laboratory and field assessment of insect growth a role in the formulation of pest management regulators for mosquito control. Mosq. News strategies in some areas (Brattsen et al. 1986, 36:462-472. Downum, K. R. and G. H. N. Towers. 1983. Analysis Smith 1976,Casida 1976, Dame et al. 1976). of thiophenes in the Tageteae () by As yet there is insufficient information to HPLC. J. Nat. Prod. 46:98-103. identify the exactchemical or chemicalsrespon- Gray, A. 1887. Gray's school and field book of botany' sible for the larvicidal activity or its mode ol' American Book Co., New York. action in the larva. In larvae exposedto lethal Hethelyi, E., P. Tetenyi, E. Dabi and B. Danos. 1987. plant levels of whole oil, the anal papillae become The role of mass spectrometry in medicinal research. Biomed. Environ. Mass. Spectrom. highly swollen, suggestingthat interruption o1' 74:627-632. osmotic and ionic regulation may be at least part Jones,T. G. H. 1926.Oleiinic terpeneketones from of the mechanism(Clements 1963). the volatiie oil of flowering T'agetesglanduLifera, Part II. J. Chem.Soc.129:2767-2770. Jones,T. G. H. and F. B. Smith. 1925.Olefinic terpene ACKNOWLEDGMENTS ketones from the volatile oil of flowering Tagetes glandulifera, Part I. J. Chem. Soc. 727:2530-2539. 1978.Isolation (Stanford Maradufu,A., R. Lubegaand F. Dorn. We are grateful to Carl Dierassi of (5E)-ocimenone,a mosquitolarvicide ftom Tag- University) and David Schooley(University of etesminuta L. Lloydia 41:181-183. Nevada at Reno) for advice during the courseof McCulloch,R. N. and D. F. Waterhouse.1947. Labo- this work and to Charles Swanson (deceased)of ratory and field tests of mosquito repellents. Aus- Potsdam, NY, and the Adirondack Museum tral. CommonwealthSci. Ind. Pes.Organ. Bull. 213. made from (BIue Mountain Lake, NY) for first suggesting Monnig, H. O. 1936.A new fly repellent (M. orl of Tagetes.Onderstepoort J. Vet. Sci. AnimaL to one of us M. G.) to look into the folklore Ind. 7:419-430. of the marigold-insect interaction. We also Naves,Y.-R. 1948.Etudes sur les matieresvegetales thank Revlon Corporation for allowing use of volatilesLVI. Sur de nouvellescetones, les tageten- equipment necessaryfor this work, David Tay- ones,isolees de I'huille essentiellede Lippia asperi' lor, for carrying out preliminary experimentson foliaRich.Helv. Chim. Acta 31:29 32. of Tagetes.Econ. the plant material treatment and Justin Skoble Neher,R. T. 1968.The ethnobotany Bot.22:317-325. preparing samplesfor the barrel tests. This for Saxena,B. P. and J. B. Srivastava.1973. Tagetes is New Jersey Agricultural Experiment Station minuta L. oil-a new sourceof juvenile hormone Publication No. D-40100-01-90,supported by mimickingsubstance. Ind. J. Exp. Biol. 11:56-58. Hatch Funds. Smith, R. F. 1976.Insecticides and integratedpest 286 JouRual oF THEArr.rpnrcar.t Mosqulro Co^rirRor,Assocrerron Vol. 7,No.2

management.pp. 489-506..In: R. L. Metcalf and J. matocidal principle isolated fromTagetes roots. Re- J. McKelvey (eds.).Future for insecticides:needs cueil 78:382-390. and prospects.Wiley, New york. deVilliers, D. J. J., C. F. Garbers and R. N. Laurie. Uhlenbroek,J. H. and J. D. Bijloo. 195g.Investiga- 1971. Synthesis of tagetenones and their occurrence tions on nematocides L lsolation and structureo-f a in oil of Tagetesminuta. Phytochem. 10:1859-1361. nematocidal principle occurring in Tagetes rcots. Weyerstahl, P., W. Zambik and C. Gansau. 1gg6. Recueil77:1004-1009. Terpene und terpen-derivate, XVIII thermische und Uhlenbroek,J. H. and J. D. Bijloo. 19b9.Investisa- AlClr-katalysierte reaction von ocimenon. Liebees tionson nematocidesIl. Structureof a secondie_ Ann. Chem. 1986:422-427.