ORIGINAL PAPER

Effect of Ageratum houstonianum Mill. (Asteraceae) leafextracts on the oviposition activity of Anopheles stephensi,Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae)

Samuel Tennyson & K. John Ravindran & Alex Eapen &S. John William

Received: 20 July 2012 /Accepted: 8 August 2012 /Published online: 19 August 2012# Springer-Verlag 2012

Abstract Plant extracts have been studied extensively fortheir insecticidal activity against immature stages and adultmosquitoes. They have also been reported to influence thehabitat preference of ovipositing mosquitoes. Ageratum hous-tonianum, a medicinal plant belonging to the family Astera-ceae, has been reported to possess insecticidal activity, and inthe present study, the ovipositional attractant/deterrent activitywas studied. The effect of Ageratum houstonianum crude leafextracts on the oviposition of Anopheles stephensi, Aedesaegypti and Culex quinquefasciatus was studied at 0.1 % con-centration in laboratory. Among hexane, ethyl acetate andmethanol crude leaf extracts, methanol showed an effectivedeterrent activity against all the three vector species with anoviposition active index of 0.8, 0.8 and 0.9, respectively.Field trials carried out to study the effect of 0.1 % methanolextract on oviposition of Aedes species indicated effectivedeterrence ranging from 79.0 to 100.0 % in indoor and 74.6to 100.0 % in outdoor ovitraps. The potential ovipositiondeterrent property of Ageratum houstonianum crude leafextracts observed in both laboratory and field studies indicatesthe presence of phytocompounds that act as effective contactdeterrent. Further, isolation, identification and preparation of

suitable formulation of the effective phytocompounds of Ager-atum houstonianum that act as a contact deterrent are required.

Introduction

Oviposition in insects differs considerably and contributesgreatly to the evolutionary success of a species. Ovipositionincluding oviposition site selection is an innate attribute fordistribution of insect species, including mosquitoes. In mos-quitoes, preference for ovipositing habitat is distinct in differ-ent species, and both abiotic (Bentley andDay 1989) and bioticcomponents (Su and Mulla 1999) of the habitat are required tobe conducive for oviposition. Research has been carried out tofind suitable tools to attract or deter ovipositing mosquitoes toaid in the control of vector mosquitoes. Plant extracts havebeen extensively screened for the same (Nathan et al. 2006a, b;Rajkumar and Jebanesan 2008; Elango et al. 2010). AgeratumhoustonianumMill. (Asteraceae), a medicinal plant reported topossess antifungal (Pandey et al. 1984), antimicrobial (Samuelet al. 2011a) and insecticidal properties (Bowers et al. 1976;Ravindran et al. 2012), is found widely distributed in India,Central America, Europe (Johnson 1971), Mexico and South-ern America (Wiedenfeld and Cetto 2001). The influence ofcrude leaf extracts of this plant on the oviposition of vectormosquitoes is reported in the present study.

Materials and methods

Preparation of plant extract

Ageratum houstonianum was collected from the foothillregions of Javadhu hills, Tiruvanamalai District, TamilNadu, India. Taxonomical identity of the plants was

S. Tennyson (*)Department of Zoology, Madras Christian College,Chennai 600 059 Tamil Nadu, Indiae-mail: samtennyson@gmail.com

K. J. Ravindran :A. EapenNational Institute of Malaria Research (ICMR) Field Unit,NIE Campus, Chennai 600 077 Tamil Nadu, India

S. J. WilliamDepartment of Advanced Zoology and Biotechnology,School of Entomology and Centre for Natural ResourcesManagement (SECNARM), Loyola College,Chennai 600 034 Tamil Nadu, India

Parasitol Res (2012) 111:22952299DOI 10.1007/s00436-012-3083-7

confirmed at the Department of Plant Biology and Biotech-nology, Loyola College, Chennai, Tamil Nadu, India. Hex-ane, ethyl acetate and methanolic crude leaf extractsobtained by sequential extraction method reported else-where (Ravindran et al. 2012) were stored at 4 C.

Laboratory bioassay

The influence of hexane, ethyl acetate and methanol crudeleaf extracts of Ageratum houstonianum on the oviposi-tion activity of vector mosquitoes was studied at 0.1 %concentration. Twenty gravid female mosquitoes werereleased into 2-ft cage and were maintained at a photope-riod of 12-h light and dark cycle. Plastic bowls (11.5 cmin diameter and 5.5 cm in height) used as ovitraps werekept inside the cage, and their position was changed in acyclic manner during each trial to avoid selection bias.The ovitraps were removed for egg count after 24 h forAnopheles stephensi and Aedes aegypti and 96 h for Culexquinquefasciatus. A total of 25 trials were carried out forassessment. Trials were repeated when eggs were not laidin any one of the ovitraps placed inside the cage. One-way analysis of variance followed by Tukeys test wasperformed to assess the effect on mosquito oviposition ontreatment with different extracts. Effective deterrence andoviposition active index (OAI) were calculated using thefollowing formula:

Effective deterrence % NC NTNC

100

where NC is the number of eggs laid in control and NT isthe number of eggs laid in treatment.

Oviposition active index NT NSNT NS

where NT is the total number of eggs laid in test solutionand NS is the total number of eggs laid in controlsolution.

Oviposition active indexes of +0.3 and above are con-sidered as attractants while those with 0.3 and below areconsidered as deterrents (Kramer and Mulla 1979). Posi-tive value indicates that more eggs were deposited intreated solutions, and negative value, more eggs incontrol.

Field bioassay

Study site

Field observations were undertaken in Pudur in thewestern outskirts of Chennai, Tamil Nadu, India. A totalof 15 houses in four streets were selected randomly forthe study.

Preparation and placement of ovitraps

In the field study, the effect of 0.1 %methanolic leaf extract onoviposition of Aedes species was studied. White plastic bowlsmeasuring 9.5 cm in diameter and 11.0 cm in height were usedas ovitraps. Outside of the bowl was painted black. A thinpaddle of wood whose length is 13.0 cm and width is 1.0 cmwas placed inside the bowl lined with a white filter paper foroviposition support. The ovitraps thus prepared were filledwith 500 ml of experimental and control solutions for the fieldstudy. One treated and one control ovitraps were placed insideeach house, and similarly one such set was kept outside. Allthe bowls inside the houses were placed in bedrooms. Mos-quito repellents were not used during the study period. Bowlsplaced outside the houses were kept under shade.

Field observations

Observations on usage/utility of the ovitraps for ovipositionby Aedes species was carried out on days 1, 2, 3, 4, 7, 14, 21and 28. During each visit, eggs/larvae present in the ovitrapswere collected and brought to the laboratory where it wasidentified and counted with a help of a dissection micro-scope. Eggs were allowed to hatch, and the larvae werereared until adult emergence for species identification. Thetreated/control solutions were replenished during each visit.Effective deterrence was calculated to assess the effect ofmethanolic leaf extract on the oviposition of Aedes mosqui-toes under field conditions.

Monitoring of natural breeding in the study site

Natural mosquito breeding status in the houses selected forthe study was recorded. The habitat-wise positivity wasnoted, and immatures were collected and brought to thelaboratory for rearing until adult emergence. This was con-tinued during every sampling occasion. Adult mosquitoesemerged were identified to understand species composition.A well net (diameter 17.0 cm) was used to sample larvaefrom wells, and a dipper/ladle (diameter 7.5 cm and capacity200 ml) was used to sample immature in cisterns, barrelsand other associated habitats in the houses.

Results

Laboratory bioassay

The study was conducted at room temperature of 26.81.7 Cand relative humidity of 77.44.2 %. During all trials, Anoph-eles stephensi, Aedes aegypti and Culex quinquefasciatus laideggs in untreated control ovitraps (water). In treated controlovitraps (Tween 80) and extract-treated ovitraps, eggs were not

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always laid. In treated control ovitraps, Culex quinquefasciatusdid not lay eggs in four trials. In hexane, ethyl acetate andmethanol extract-treated ovitraps, Anopheles stephensi did notlay eggs in 2, 2 and 4 trials; Aedes aegypti in 1, 1 and 1; andCulex quinquefasciatus in 13, 19 and 21 trials, respectively.

The total egg count in all ovitraps (extract treated + treatedcontrol + untreated control) varied considerably in all trialsand ranged from 218 to 469 in Anopheles stephensi, 104 to596 in Aedes aegypti and 4 to 19 rafts in Culex quinquefas-ciatus. The minimum and maximum number of eggs laid byAnopheles stephensi in treated and untreated control ovitrapswas 25.8 and 49.3, 34.4 and 62.2; by Aedes aegypti, it was 1.8and 39.2, 34.4 and 71.0; and by Culex quinquefasciatus, 0.0and 57.1, 28.6 and 100.0, respectively. In hexane, ethyl acetateand methanol extract-treated ovitraps, it was 0.0 and 17.3, 0.0and 12.6, 0.0 and 9.8; 0.0 and 17.3, 0.0 and 33.6, 0.0 and 10.7;0.0 and 20.0, 0.0 and 28.6, 0.0 and 16.7, respectively.

The percentage of eggs laid in hexane, ethyl acetate andmethanol extracts to the eggs laid in all ovitraps was below

10 % in 12, 16 and 25 of the total trials in Anophelesstephensi; 19, 11 and 24 in Aedes aegypti; 15, 20 and 22in Culex quinquefasciatus, respectively. The OAI rangedbetween 0.5 and 0.9 among the extracts for all the vectorspecies studied (Table 1).

Field bioassay

Immatures of Aedes species were found in both householdhabitats and the ovitraps placed. Out of the 14 wells, fiveoverhead tanks and five cisterns checked regularly, imma-ture of Aedes species was obtained only in the cisterns andhabitat positivity ranged from 0 to 33.3 %. Aedes specieslaid eggs in ovitraps kept indoors and outdoors. During theprefixed 8 days of observation, Aedes breeding was noted intreated and control indoor ovitraps for 3 and 6 days and 3and 5 days in outdoor ovitraps. Ovitrap positivity in treatedand control indoor ovitraps ranged from 6.7 to 7.1 and 6.7 to40.0 %. Mean number of eggs laid ranged from 0.10.3 to

Table 1 Influence of the crude extracts of Ageratum houstonianum leaves on the ovipositional activity of vector mosquitoes

Particulars Vector mosquito species Hexane Ethyl acetate Methanol Treated control (Tween 80) Untreated control

Number of eggs laid Anopheles stephensi 33.815.2 a 28.413.1 a 15.98.1 a 111.723.5 b 154.945.1 c

Aedes aegypti 23.016.0 a 54.837.4 b 16.411.9 a 94.954.9 c 184.664.5 d

Culex quinquefasciatus 0.60.8 a 0.30.6 a 0.20.4 a 2.31.9 b 5.23.0 c

ED (%) Anopheles stephensi 78.2 81.7 89.7 27.9 NA

Aedes aegypti 87.5 70.3 91.1 48.6 NA

Culex quinquefasciatus 88.5 93.9 96.9 56.6 NA

OAI Anopheles stephensi 0.6 0.7 0.8 0.2 NA

Aedes aegypti 0.8 0.5 0.8 0.3 NA

Culex quinquefasciatus 0.8 0.9 0.9 0.4 NA

n025. Different lowercase letters in the same row of different extracts show significant difference at P

0.93.4 and 0.62.3 to 9.715.4, respectively (Fig. 1). Inoutdoors, ovitrap positivity was 6.7 to 20.0 and 6.7 to100.0 %, respectively, and the mean number of eggs laidranged from 1.14.4 to 4.39.7 and 1.76.5 to 57.681.6,respectively (Fig. 2). Effective deterrence in ovipositionindoors and outdoors ranged from 79.0 to 100.0 and 74.6to 100.0. The mode effective deterrence was 100.0 % inindoors and outdoors. Mosquito immatures that emergedfrom these ovitraps collections were all Aedes aegypti.

Discussion

Plant extracts have been studied extensively for their insecti-cidal property against immature (Sakthivadivel and Daniel2008; Arivoli and Samuel 2011; Samuel et al. 2011b; Arivoliet al. 2012) and adult (Choochote et al. 2006; Senthilkumar etal. 2009; Ravindran et al. 2012) mosquitoes. Comparatively,there are few reports on the influence of plant extracts on theoviposition of mosquitoes (Elango et al. 2010). There are noreports on the effect of Ageratum houstonianum on oviposi-tion of mosquitoes. Results of the present study indicated thatleaf extracts of Ageratum houstonianum influenced oviposi-tion of vector mosquitoes.

Ovitrap positivity for Anopheles stephensi, Aedes aegyptiand Culex quinquefasciatus breeding in laboratory trials indi-cated preference of the vector mosquitoes to oviposit in allovitraps (extract-treated, treated control and untreated con-trol). This result shows that the adult ovipositing mosquitowas not repelled by extracts. However, when taking intoconsideration the number of eggs laid, few eggs were laid inextract-treated ovitraps when compared to control indicatingthat oviposition was deterred after coming in contact with thewater treated with crude leaf extracts of Ageratum houstonia-num. Further, oviposition active index ofAnopheles stephensi,Aedes aegypti and Culex quinquefasciatus was below 0.3confirming the deterrent nature (Kramer and Mulla 1979) ofleaf extract. Among the three extracts, methanol was highlydeterrent against all three vector species. The ovipositiondeterrent activity of methanolic leaf extract of Ageratumhoustonianum was comparatively higher to methanolic leafextracts of Pelargonium citrosa (Jeyabalan et al. 2003) andDysoxylum malabaricum against Anopheles stephensi(Nathan et al. 2006b). Methanolic leaf extract of the formershowed 56 % effective deterrence at a concentration of 1 %and the latter showed an OAI of 0.5 at 0.5 %. Other plantextracts have also been reported to exhibit oviposition deter-rent activity against Aedes aegypti mosquitoes (Angerilli1980; Judd and Borden 1980; Sharma et al. 1981) Field trialswith methanolic extracts also showed high deterrence to ovi-positing Aedes aegypti mosquitoes. Effective deterrence was100 % in most of the days of observation in both indoor andoutdoor ovitraps, manifesting strong deterrent characteristics

of the extract. This oviposition deterrent property can beexploited to prevent Aedes aegypti breeding in temporary/seasonal non-potable habitats.

Oviposition pheromones, kairomones (Takken and Knols1999) and organic compounds (Ikeshoji et al. 1975; Millaret al. 1992; Mordue et al. 1992; Beehler et al. 1994; Mboeraet al. 1999) have been reported to influence the attractive-ness of the ovipositing site. Plant extracts have also beenstudied for the same (Rajkumar and Jebanesan 2005; Elangoet al. 2010). The potential oviposition deterrent property ofAgeratum houstonianum crude leaf extracts observed inboth laboratory and field studies indicates the presence ofphytocompounds that act as an effective contact deterrent.Further, isolation, identification and preparation of suitableformulation of the effective phytocompounds of Ageratumhoustonianum that act as a contact deterrent are required.

Acknowledgments Authors are thankful to the staff of the NationalInstitute of Malaria Research (ICMR), Field Unit, Chennai, TamilNadu, India for their kind assistance.

References

Angerilli NPD (1980) Influences of extracts of freshwater vegetationon the survival and oviposition by Aedes aegypti. Can Entomol112:12491252

Arivoli S, Samuel T (2011) Larvicidal efficacy of Cleistanthus collinus(Roxb.) (Euphorbiaceae) leaf extracts against vector mosquitoes(Diptera: Culicidae). Asian Pac J. Trop Biomed 1(2):S275S277

Arivoli S, Ravindran J, Samuel T (2012) Larvicidal efficacy of plantextracts against the malarial vector Anopheles stephensi Liston(Diptera: Culicidae). World J Med Sci 7(2):7780

Beehler JW, Millar JG, Mulla MS (1994) Field evaluation of syntheticcompounds mediating oviposition in Culex mosquitoes (Diptera:Culicidae). J Chem Ecol 20:281291

Bentley MD, Day JF (1989) Chemical ecology and behavioural aspectsof mosquito oviposition. Annu Rev Entomol 34:401421

Bowers WS, Ohta T, Cleeve JS (1976) Discovery of insect antijuvenilehormones in plants. Science 193:542547

Choochote W, Chaithong U, Kamsuk K, Rattanachanpichai E, JitpakdiA, Tippawangkosol P, Chaiyasit D, Champakaew D, Tuetun B,Pitasawat B (2006) Adulticidal activity against Stegomyia aegypti(Diptera: Culicidae) of three Piper spp. Rev Inst Med Trop SaoPaulo 48(1):3337

Elango G, Rahuman AA, Bagwan A, Kamraj C, Zahir AA, RajakumarG, Marimuthu S, Santhoshkumar T (2010) Efficacy of botanicalextracts against Japanese encephalitis vector, Culex tritaeniorhyn-chus. Parasitol Res 106:481492

Ikeshoji T, Saito K, Yano A (1975) Bacterial production of the ovipo-sitional attractants for mosquitoes on fatty acid substrates. ApplEntomol Zool 10:239242

Jeyabalan D, Arul N, Thangamathi P (2003) Studies on the effects ofPelargonium citrosa leaf extracts on malaria vector, Anophelesstephensi Liston. Biores Technol 89:185189

JohnsonMF (1971) Amonograph of the genus AgeratumL. (Compositae-Eupatorieae). Ann Mo Bot Gard 58:688

Judd JGR, Borden JH (1980) Oviposition deterrents for Aedes aegyptiin extracts of Lemna minor. J Entomol Soc 17:3033

2298 Parasitol Res (2012) 111:22952299

Kramer WL, Mulla S (1979) Oviposition attractants and repellents ofmosquitoes: oviposition responses of Culex mosquitoes to organicinfusions. Environ Entomol 8:11111117

Mboera LEG, Midra KY, Salum FM, Takken W, Pickett JA (1999)The influence of synthetic oviposition pheromone and volatilesfrom soakage pits and grass infusions upon oviposition siteselection of Culex mosquitoes in Tanzania. J Chem Ecol25:18551865

Millar JG, Chaney JD, Mulla MS (1992) Identification of ovipositionattractants for Culex quinquefasciatus from fermented Bermudagrass infusions. J Am Mos Contr Assoc 8:1117

Mordue AJ, Blackwell A, Hansson BS, Wadhams LJ, Pickett JA(1992) Behavioural and electrophysiological evaluation of oviposi-tion attractants for Culex quinquefasciatus Say (Diptera: Culicidae).Experientia 48:11091111

Nathan SS, Kalaivani K, Sehoon K (2006a) Effects of Dysoxylummalabaricum Bedd. (Meliaceae) extract on the malaria vectorAnopheles stephensi Liston (Diptera: Culicidae). Biores Technol97:20772083

Nathan SS, Savitha G, George DK, Narmadha A, Suganya L, ChungPG (2006b) Efficacy ofMelia azedarach L. extract on the malariavector Anopheles stephensi Liston (Diptera: Culicidae). BioresTechnol 97:13161323

Pandey DK, Chandra H, Tripathi NN, Dixit SN (1984) Myco-toxicity in leaves of some higher plants with special refer-ence to that of Ageratum houstonianum Mill. Mykosen26:565573

Rajkumar S, Jebanesan A (2005) Oviposition deterrent and skin repellentactivities of Solanum trilobatum leaf extract against the malariavector Anopheles stephensi. J Insect Sci 5(15):13

Rajkumar S, Jebanesan A (2008) Bioactivity of flavonoid compoundsfrom Poncirus trifoliata L. (Family: Rutaceae) against the denguevector Aedes aegypti (Diptera: Culicidae). Parasitol Res 104:1925

Ravindran J, Samuel T, Alex E, William J (2012) Adulticidal activityof Ageratum houstonianum Mill. (Asteraceae) leaf extractsagainst three vector mosquito species (Diptera: Culicidae). AsianPac J Trop Dis 2(3):177179

Sakthivadivel M, Daniel T (2008) Evaluation of certain insecticidalplants for the control of vector mosquitoes viz., Culex quinque-fasciatus, Anopheles stephensi and Aedes aegypti. Appl EntomolZool 43(1):5763

Samuel T, Balaraju K, Kyungseok P, Raja AK, Ravindran KJ, Alex E,William SJ (2011a) In vitro antimicrobial activity of Ageratumhoustonianum Mill. (Asteraceae). Food Sci 35:28972900

Samuel T, Ravindran J, Arivoli S (2011b) Screening of plant extractsfor ovicidal activity against Culex quinquefasciatus Say (Diptera:Culicidae). Appl Bot 40:54565460

Senthilkumar N, Varma P, Gurusubramanian G (2009) Larvicidal andadulticidal activities of some medicinal plants against the malariavector, Anopheles stephensi (Liston). Parasitol Res 104:237244

Sharma RN, Joshi V, Jadu G, Bhosale AS, Gupta AS (1981) Oviposi-tion deterrence activity in some Lamiaceae plants against insectpests. Z Naturforsh Teil C 36:122125

Su T, Mulla MS (1999) Ovipositional bioassay responses of Culextarsalis and Culex quinquefasciatus to neem products containingazadirachtin. Entomol Exp Appl 91:337345

Takken W, Knols BGJ (1999) Odor mediated behaviour of afrotropicalmalaria mosquitoes. Annu Rev Entomol 44:131157

Wiedenfeld H, Cetto AA (2001) Pyrrolizidine alkaloids from Ageratumhoustonianum Mill. Phytochem 57:12691271

Parasitol Res (2012) 111:22952299 2299

Effect...AbstractIntroductionMaterials and methodsPreparation of plant extractLaboratory bioassayField bioassayStudy sitePreparation and placement of ovitrapsField observationsMonitoring of natural breeding in the study site

ResultsLaboratory bioassayField bioassay

DiscussionReferences