H. Charulatha

K. Prabakaran

K. Vasudevan

R. Malarmagal

Venkata Lakshmi Saraswatula

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J Vector Borne Dis 46, June 2009, pp. 153–156Short Research CommunicationsLarvicidal effects of crude extracts of dried ripened fruits ofPiper nigrum against Culex quinquefasciatus larval instarsK. Vasudevan, R. Malarmagal, H. Charulatha, Venkata Lakshmi Saraswatula &K. PrabakaranDepartment of Zoology, Annamalai University, Annamalai Nagar,  Tamil Nadu, IndiaKey words    Culex quinquefasciatus – dried ripened fruits – larvicidal – Piper nigrumThe medical importance of mosquitoes as vectors fortransmission of serious diseases that cause morbid-ity, mortality, economic loss, and social disruptionsuch as malaria, lymphatic filariasis, and viral dis-eases is well-documented1. Mosquitoes not onlycause nuisance by their bites but also transmit deadlydisease like malaria, filariasis, yellow fever, dengueand Japanese encephalitis, contribute significantly topoverty and social debility in tropical countries2. Themosquito Culex quinquefasciatus act as a vector forWuchereria bancrofti responsible for filariasis inIndia. However, control of such diseases are becom-ing increasingly difficult because the overproductionof detoxifying mechanisms of chemical insecticideshas been reported for Culex species3.Humans have used plant parts, products and metabo-lites in pest control since early historical times. Plantsare the chemical factories of nature, producing manychemicals, some of which have medicinal and pes-ticidal properties. By using plant parts in early his-torical times and plant extracts and concentratedcomponents in more recent times, man has been ableto control certain pests with these remedies quitesuccessfully. The current use and future potential ofplants for pest control on farms and homes are de-tailed in an FAO document4.  Sukumar et al5 pub-lished an extensive review of phytochemicals fromplants with activity against mosquitoes. Members ofthe plant families—Asteraceae, Cladophoraceae,Labiateas, Miliaceae, Oocystaceae and Rutaceaepossess various types of activity against many spe-cies of mosquitoes5. Wood6 lists some importantphotochemical products such as pyrethrum, derris,quassia, nicotine, hellebore, anabasine, azadirachtin,d-limonene, camphor and terpenes that have beenused as insecticides. These are major groups of insec-ticides of plant origin that were used in developedcountries before the advent of synthetic organic in-secticides7. Ethanolic extract of Centella asiaticaleaves is promising as larvicide and adult emergenceinhibitor against Cx. quinquefasciatus larvae8.More than 2000 plant species have been known toproduce chemical factors and metabolites of value inpest control programmes9. One of the most studiedbotanical species with good pesticidal attributes, isthe neem tree (Azadirachta indica A. Juss), whoseextracts have shown considerable activity and mul-tiple modes of action against agricultural pests, for-estry insects, and insects of public health10,11.Larvicide constituents from Lanta viburnodis sp havereported by Innocent et al12. However, studiesagainst the pest of public health importance are to-tally lacking. In the present study, the extracts ofdried fruits of Piper nigrum have been evaluatedagainst the larvae of important vector of filariasis andthe findings are summarised here.For evaluating larvicidal activity of Piper nigrum, J  VECTOR  BORNE  DIS  46, JUNE 2009 154dried ripened fruits were obtained from Tamil NaduAgricultural Cooperative Society, a voucher speci-men deposited at the Department of Botany,Annamalai University. Aqueous (PnAE) andethanolic extracts (PnEE) of P. nigrum were used inthe experiments. Each one kg of thoroughly cleanedair-dried in the shade, powdered material of P.nigrum was soaked in 3 L of double distilled water(PnAE) and 95% ethanol (PnEE) at room tempera-ture for 48 h was suction filtered through a Buchnerfunnel and concentrated to dryness with rotaryevaporator at 60°C, until the solvents completelyevaporated. The aqueous (PnAE) and ethanolic(PnEE) extract was thus obtained, lyophilised andthen refrigerated at –20°C until testing for larval in-stars toxicities.The test organism, namely Cx. quinquefasciatus lar-vae, which were derived from the Medical Ento-mology Field Station (ICMR), Vriddhachalam andVector Control Research Centre (ICMR), Puducherrywhere colonized and maintained in a laboratory freeof exposure to pathogens, insecticides or repellents.The laboratory colony was maintained at 25–30°Cand 80–97% relative humidity under a photoperiodof 14:10 h (light/dark) in the insectary, Faculty ofScience, Annamalai University, Annamalai Nagar.Under these conditions, the full developmentfrom egg to adult lasted about three weeks. Larvaewere fed on finely ground dog biscuits. Theadult colony was provided with 10% sucrose and10% multivitamin syrup and it was periodicallyblood-fed on restrained rabbits. Four developmen-tal stages of larvae were available for the experi-ments. Under these conditions the life cycle ofthe mosquito from egg to adult took about 3–4weeks. Early IV instar larvae were used in the ex-periments.The larvicidal bioassay followed the WHO standardprotocols13 with slight modifications. Each pepperextract (PnAE and PnEE) was dissolved in absoluteethanol to prepare graded series of concentration.The stock solution (500 mg/ml) of aqueous (PnAE)and ethanolic extract (PnEE) of P. nigrum was volu-metrically diluted to 250 ml filtered tap water to ob-tain the test solutions of 20, 40, 60, 80, 100 and 120mg/l for PnAE; 5, 10, 20, 40, 60, 80, and 100 mg/lof PnEE in a 500 ml enamel bowl, which was shakenlightly to ensure a homogenous solution. Then 20early IV instar larvae of the mosquito in each 25 mlof distilled water were transferred to that bowl. Eachexperiment was performed in six replicates for eachconcentration.Symptoms of treated larvae were observed and re-corded immediately and at time intervals, and nofood was offered to the larvae. Mortality and survivalwere registered after 24 h of exposure period. Themoribund and dead larvae in six replicates were com-bined and expressed as a percentage of larval mor-tality of each concentration. Dead larvae wereidentified when they failed to move after probingwith needle in the siphon or cervical region. Mori-bund larvae were those incapable of riding to thesurface. They might also show discolouration un-natural positions, tremors, uncoordination or rigor.The experiments were replicated for six times.Larvicidal activity of PnAE and PnEE against earlyIV instars of the mosquito were registered. In casescontrol mortality, observed mortality was correctedby using Abbott’s formula14. LC50 and LC90 confi-dence intervals were analysed by means of comput-erized probit analysis (SPSS 12.0), yielding a levelof effectiveness at 50 and 90% mortality, and 95%confidence interval were used to measure differencesbetween test samples.Aqueous (PnAE) and ethanolic (PnEE) extracts ofP. nigrum with a yield of 2.18% (w/w) and 3.24%(w/w), were semisolid, light yellow brownish andstrong aromatic. Larvicidal activity of PnAE againstearly IV instar larvae of Cx. quinquefasciatus isshown in Table 1.In this study, early IV instar larvae of Cx. quinque-fasciatus, under laboratory conditions, were sub-jected to a dose-dependent efficacy of P. nigrum(PnAE and PnEE) extracts. 155 VASUDEVAN ET AL: LARVICIDAL EFFECTS OF PIPER NIGRUMAqueous extracts of P. nigrum (PnAE) caused mor-tality against early IV larval instars of Cx.quinquefasciatus. However, the sensitivity of larvalinstars was positively correlated dose-dependentlevel with PnAE (Table 1). Following the treatmentof increasing dosages of PnAE from 20–120 mg/l.The 24 h LC50 and LC90 against early IV larval in-stars were 63.82 and 108.90 mg/l, respectively. Theresults showed that this extract PnAE can be used tocontrol larval instars of the mosquitoes. Ethanolicextracts of P. nigrum (PnEE), observations on theresults of the larval susceptibility were given in Table1. With the IV instars of the mosquito species, mor-tality was dose-dependent—mortality increased withincrease in concentration of PnEE. LC50 and LC90values along with their fiducial limits, regressionequation and chi-square were calculated. LC50 andLC90 values as observed for early  IV larval instarsof Cx. quinquefasciatus were 29.11 and 62.37 mg/l,respectively (Table 1). Results of the experiments en-visaged larvicidal property in both PnAE and PnEEextracts of P. nigrum. As the dried fruits of P. nigrumare available most of the time throughout the coun-try, the larvicidal properties of the plant species canbe well-utilized, while planning alternate vector con-trol programmes.Today, the environmental safety is considered to beof paramount importance. An insecticide does nothave to cause high mortality on target organisms inorder to be acceptable14. Phytochemicals may serveas they are relatively safe inexpensive, and are readilyavailable in many parts of the world. According toBowers et al15, the screening of locally availablemedicinal plants for mosquito control would gener-ate local employment, reduce dependence on expen-sive and imported products, and to stimulate localefforts to enhance public health system.The crude extracts of dried fruits of P. nigrum bothaqueous (PnAE) and ethanolic (PnEE) have beenfound to possess larvicidal effect against the IV lar-val instars of the mosquito Cx. quinquefasciatus. Thebiological activity of the PnAE and PnEE might bedue to the various compounds including potentiallyinsecticidal compounds16,17. Some of the piper spe-cies,  P. longum, P. guanacastensis and theirbioactive constituents are reported to have remark-able larvicidal activity against various mosquito spe-cies such as Cx. pipiens pallens, Ae. aegypti, Ae.togoi and Ae. atropalpus18,19. In this study, early IVinstar larvae of Cx. quinquefasciatus under labora-tory conditions, were subjected to a dose-dependentefficacy of the two piper extracts (Table 1). Follow-ing the treatment of increasing the dosages of PnAEand PnEE of P. nigrum, the larval mortality increasedsignificantly as complete mortality occurred. In thecase of the control or untreated group mortality ob-served within 24 h was within admissible range andthe IV larvae development into pupae and then toadults with 48–72 h.Among the extracts, PnEE showed more remarkablelarvicidal potency than PnAE. Variety of types andlevels of active constituents  (pipernoneline and pi-peridine alkaloid)18, (methly 4-hydroxyl-3-(3’-me-thyl 2’ butenyl) benzoate)19 in the piper species maybe responsible for the variability in their potentialagainst Cx. quinquefasciatus larvae.Although the larvicidal potential of crude aqueousextracts derived from P. nigrum dried fruits observedTable 1. Larvicidal activities of  extracts of P. nigrum against early IV larval instars of Cx. quinquefasciatusType of LC50 mg/l 95% confidence LC90 mg/l 95% confidence Regression χ2extract  limit (mg/l)  (in water) limit (mg/l) equationAqueous 63.82 59.81–67.90 108.90 102.47–116.78 Y = 47.057+0.823 x 10.426*Ethanolic 29.11 22.25–36.50 62.37 52.21–79.16 Y = 19.213+0.978 x 22.143†*p < 0.05; †p <0.001; Each value (mean ± S.D.) represents mean of six values. J  VECTOR  BORNE  DIS  46, JUNE 2009 156in the study was significantly lower than that ofethanolic extracts (PnEE) further studies for the iso-lation and identification of bioactive compoundswould be useful in developing new types of mosquitolarvicides. However, this study with PnAE and PnEEdemonstrated the potential of P. nigrum extractsagainst IV instar larvae of Cx. quinquefasciatus andits benefits to developing cost-effective and, environ-ment friendly new type of larvicide for mosquitocontrol needs to be studied.AcknowledgementThe authors acknowledge the staff members of theVCRC (ICMR), Puducherry for their cooperation.We thank Prof. M. Sabesan, Professor and WingHead, for his extended cooperation for the laboratoryfacilities and support in publishing this paper.References1. Becker N, Petriae D, Zgomba M, Boase C, Dahl C, LaneJ, Kaiser A. Mosquitoes and their control. New York:Kluwer Academic Plenum Publisher 2003; p. 1–16.2. Jang YS, Kim MK, Ahn YJ, Lee HS. Larvicidal activityof Brazilian plants against Aedes aegypti and Culex pipienspallen (Diptera : Culicidae). Agric Chem Biotechnol 2002;45(3): 131–4.3. Severini C, Rom R, Marinucci M, Rajmond M. Mecha-nisms of insecticide resistance in field population of Culexpipiens from Italy. J Am Mosq Control Assoc 1993; 9:164–8.4. Use and potential of wild plants in farm house holds. FAOInformation Division. Food and Agricultural Organiza-tion of the United Nations 1999. Available from: http://www.fao.org/docrep/003/w8801eoo.htm.5. Sukumar K, Perich MJ, Booba LR. Botanical derivativesin mosquito control : a review. J Am Mosq Control Assoc1991; 7: 210–37.6. Wood A. Compendium of pesticide common names—insecticides 2003.  Available from: http://www.alanwood.net/pesticides/index.htm.7. Casida JE, Quistad GB. Golden age of insecticide research:past, present or Future? Anu Rev Entomol 1998; 43: 1– 16.8. Rajkumar S, Jebanesan A. 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JVector Borne Dis 2008; 45: 240–4.13. Instructions for determining the susceptibility or resis-tance of mosquito larvae to insecticides. WHO/VBC/81.807.14. Abbott WS. A method of computing the effectiveness ofan insecticide. J Econ Entomol 1925; 18: 265–6.15. Bowers WS, Sener B, Evans PH, Bingol F, Erdogan I.Activity of Turkish medicinal plants against mosquitoesAedes aegypti and Anopheles gambiae. Insect Sci Appl1995; 16(34): 339–42.16. Freeborn SB, Wymore FH. Attempts to protect sweet cornfrom infestations of the corn earworm, Heliothis obsoleta(Fabr). J Econ Entomol 1929; 22: 666–71.17. Su HCF, Horvat R. Isolation, identification and insecti-cidal properties of Piper nigrum amides. J Agric FoodChem 1981; 29: 115–8.18. Lee SE. Mosquito larvicidal activity of pipernonaline, apiperidine alkaloid derived from long pepper, Piperlongum. J Am Mosq Control Assoc 2000; 16: 245–7.19. Pereda-Miranda R, Bernard CB, Durst T, Arnason JT,Sanchez-Vindes, et al. Methyl 4-hydroxyl-3-(3’-Methyl-2’-butenyl) benzoate, major insecticidal principle fromPiper guanacastensis. J Wat Prod 1997; 60: 282–4.Corresponding author: Dr K. Vasudevan, Reader in Zoology, Department of Zoology (DDE),  Annamalai University,Annamalai Nagar–608 002, India.E-mail: drkvasudevan@yahoo.comReceived: 4 December 2008 Accepted in revised form: 4 February 2009

Larvicidal effects of crude extracts of dried ripened fruits of Piper nigrum against Culex quinquefasciatus larval instars

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