ORIGINAL PAPER Larvicidal, ovicidal, and adulticidal efficacy of Erythrina indica (Lam.) (Family: Fabaceae) against Anopheles stephensi , Aedes aegypti , and Culex quinquefasciatus (Diptera: Culicidae) Marimuthu Govindarajan & Rajamohan Sivakumar Received: 7 November 2013 /Accepted: 17 November 2013 /Published online: 10 December 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Mosquitoes are the major vector for the transmis- sion of malaria, dengue fever, yellow fever, filariasis, schisto- somiasis, and Japanese encephalitis. Mosquito control is fac- ing a threat because of the emergence of resistance to synthetic insecticides. Insecticides of botanical origin may serve as suitable alternative biocontrol techniques in the future. In view of the recently increased interest in developing plant origin insecticides as an alternative to chemical insecticide, this study was undertaken to assess the larvicidal, ovicidal, and adulticidal potential of the crude hexane, benzene, chloro- form, ethyl acetate, and methanol solvent extracts from the medicinal plant Erythrina indica against the medically impor- tant mosquito vectors, Anopheles stephensi , Aedes aegypti , and Culex quinquefasciatus (Diptera: Culicidae). The larval mortality was observed after 24 h of exposure. All extracts showed moderate larvicidal effects; however, the highest lar- val mortality was found in methanol extract of leaf of E. indica against the larvae of A. stephensi , A. aegypti , and C. quinquefasciatus with the LC 50 and LC 90 values of 69.43, 75.13, and 91.41 ppm and 125.49, 134.31, and 167.14 ppm, respectively. The mean percent hatchability of the eggs was observed after 48 h post treatment. The percent hatchability was inversely proportional to the concentration of extract and directly proportional to the eggs. All the five solvent extracts showed moderate ovicidal activity; however, the methanol extract showed the highest ovicidal activity. The methanol extract of E. indica against A. stephensi , A. aegypti , and C. quinquefasciatus exerted 100 % mortality (zero hatchability) at 150, 200, and 250 ppm, respectively. Control eggs showed above 99.3–100 % hatchability. The adult mortality was observed after 24 h recovery period. The plant crude extracts showed dose-dependent mortality. At higher concentrations, the adult showed restless movement for some times with abnormal wagging and then died. Among the extracts tested, the highest adulticidal activity was observed in methanol extract against A. stephensi followed by A. aegypti and C. quinquefasciatus with the LD 50 and LD 90 values of 88.76, 94.09, and 119.64 ppm and 160.83, 169.01, and 219.77 ppm, respectively. No mortality was recorded in the control. Our data suggest that the crude hexane, benzene, chloroform, ethyl acetate, and methanol solvent extracts of E. indica have the potential to be used as an eco-friendly approach for the control of the A. stephensi , A. aegypti , and C. quinquefasciatus . Introduction Mosquito-borne diseases, like malaria, yellow, and dengue fevers, are a major threat to over two billion people in the tropics. Mosquito bites may also cause allergic responses including local skin reactions and systemic reactions such as urticaria and angioedema (Peng et al. 2004). Anopheles stephensi L. is the primary vector of malaria in India and other West Asian countries, and improved methods of control are urgently needed (Burfield and Reekie 2005; Mittal et al. 2005). Malaria infects more than 500 million humans each year, killing approximately 1.2 to 2.7 million/year. About 90 % of all malaria cases occur in Africa, as does approxi- mately 90 % of the world’ s malaria-related deaths (Breman et al. 2004). Malaria, caused by Plasmodium falciparum , is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub- tropical countries (Snow et al. 2005). Culex quinquefasciatus is a predominant house-resting mosquito in many tropical countries. It is important as a vector of filariasis in some countries as well as a nuisance mosquito. Mosquitoes breed M. Govindarajan (*) : R. Sivakumar Unit of Vector Biology and Phytochemistry, Department of Zoology, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India e-mail: [email protected]Parasitol Res (2014) 113:777–791 DOI 10.1007/s00436-013-3709-4
Transcript
ORIGINAL PAPER
Larvicidal, ovicidal, and adulticidal efficacy of Erythrina indica (Lam.)(Family: Fabaceae) against Anopheles stephensi , Aedes aegypti ,and Culex quinquefasciatus (Diptera: Culicidae)
Marimuthu Govindarajan & Rajamohan Sivakumar
Received: 7 November 2013 /Accepted: 17 November 2013 /Published online: 10 December 2013# Springer-Verlag Berlin Heidelberg 2013
Abstract Mosquitoes are the major vector for the transmis-sion of malaria, dengue fever, yellow fever, filariasis, schisto-somiasis, and Japanese encephalitis. Mosquito control is fac-ing a threat because of the emergence of resistance to syntheticinsecticides. Insecticides of botanical origin may serve assuitable alternative biocontrol techniques in the future. In viewof the recently increased interest in developing plant origininsecticides as an alternative to chemical insecticide, this studywas undertaken to assess the larvicidal, ovicidal, andadulticidal potential of the crude hexane, benzene, chloro-form, ethyl acetate, and methanol solvent extracts from themedicinal plant Erythrina indica against the medically impor-tant mosquito vectors, Anopheles stephensi , Aedes aegypti ,and Culex quinquefasciatus (Diptera: Culicidae). The larvalmortality was observed after 24 h of exposure. All extractsshowed moderate larvicidal effects; however, the highest lar-val mortality was found in methanol extract of leaf of E.indica against the larvae of A. stephensi , A. aegypti , and C.quinquefasciatus with the LC50 and LC90 values of 69.43,75.13, and 91.41 ppm and 125.49, 134.31, and 167.14 ppm,respectively. The mean percent hatchability of the eggs wasobserved after 48 h post treatment. The percent hatchabilitywas inversely proportional to the concentration of extract anddirectly proportional to the eggs. All the five solvent extractsshowed moderate ovicidal activity; however, the methanolextract showed the highest ovicidal activity. The methanolextract of E. indica against A. stephensi , A. aegypti , and C.quinquefasciatus exerted 100 % mortality (zero hatchability)at 150, 200, and 250 ppm, respectively. Control eggs showedabove 99.3–100 % hatchability. The adult mortality was
observed after 24 h recovery period. The plant crude extractsshowed dose-dependent mortality. At higher concentrations,the adult showed restless movement for some times withabnormal wagging and then died. Among the extracts tested,the highest adulticidal activity was observed in methanolextract against A. stephensi followed by A. aegypti and C.quinquefasciatus with the LD50 and LD90 values of 88.76,94.09, and 119.64 ppm and 160.83, 169.01, and 219.77 ppm,respectively. No mortality was recorded in the control. Ourdata suggest that the crude hexane, benzene, chloroform, ethylacetate, and methanol solvent extracts of E. indica have thepotential to be used as an eco-friendly approach for the controlof the A. stephensi , A. aegypti , and C. quinquefasciatus .
Introduction
Mosquito-borne diseases, like malaria, yellow, and denguefevers, are a major threat to over two billion people in thetropics. Mosquito bites may also cause allergic responsesincluding local skin reactions and systemic reactions such asurticaria and angioedema (Peng et al. 2004). Anophelesstephensi L. is the primary vector of malaria in India andother West Asian countries, and improved methods of controlare urgently needed (Burfield and Reekie 2005; Mittal et al.2005). Malaria infects more than 500 million humans eachyear, killing approximately 1.2 to 2.7 million/year. About90 % of all malaria cases occur in Africa, as does approxi-mately 90 % of the world’s malaria-related deaths (Bremanet al. 2004). Malaria, caused by Plasmodium falciparum , isone of the leading causes of human morbidity and mortalityfrom infectious diseases, predominantly in tropical and sub-tropical countries (Snow et al. 2005). Culex quinquefasciatusis a predominant house-resting mosquito in many tropicalcountries. It is important as a vector of filariasis in somecountries as well as a nuisance mosquito. Mosquitoes breed
M. Govindarajan (*) :R. SivakumarUnit of Vector Biology and Phytochemistry, Department of Zoology,Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, Indiae-mail: [email protected]
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in polluted waters such as blocked drains, damaged septictanks, or soak age pools close to human habitations.Lymphatic filariasis is probably the fastest spreading insect-borne disease of man in the tropics, affecting about 146million people (WHO 1992). C. quinquefasciatus is the mostwidely distributed mosquito in India, mainly found in urbanand suburban areas. The most efficient approach to controlthe vector is to target the immature stages of the life cycle.Lymphatic filariasis is a mosquito-borne disease causedby mosquito-transmitted filarial nematodes, includingWuchereria bancrofti and Brugia malayi . The infectedpeople carry the nocturnally periodic W. bancrofti , whichhas C. quinquefasciatus as the main mosquito vector. C.quinquefasciatus is a vector of lymphatic filariasis, whichis a widely distributed tropical disease with around 120million people infected worldwide, and 44 million peoplehave common chronic manifestation (Bernhard et al.2003). According to WHO, about 90 million peopleworldwide are infected with W. bancrofti , the lymphaticdwelling parasite, and ten times more people at the risk ofbeing infected. In India alone, 25 million people harbormicrofilaria and 19 million people suffer from filarialdisease manifestations (NICD 1990; Maheswaran et al.2008). Dengue is a vector-borne disease of tropical andsubtropical human populations, which occurs predomi-nantly in urban areas. Dengue is transmitted by Aedesmosquitoes that breed in container habitats. The mainvector Aedes aegypti is a cosmotropical species that pro-liferates in water containers in and around houses.Secondary vectors include Aedes albopictus , an importantvector in Southeast Asia and that has spread to theAmericas, western Africa, and the Mediterranean rim;Aedes mediovittatus in the Caribbean; and Aedespolynesiensis and Aedes scutellaris in the westernPacific region. A. aegypti breeds in many types of house-hold containers, such as water storage jars, drums, tanks,and plant or flower containers (Muir and Kay 1998;Honorio et al. 2003).
Vector control is of serious concern in developing countrieslike India due to the lack of general awareness, developmentof resistance, and socioeconomic reasons. Every year, a largepart of the population is affected by one or more vector-bornediseases. Vector control, which includes both antilarval andantiadult measures, constitutes an important aspect of anymosquito control programs. Control either by biological orchemical means is the basic requirement for planning aneffective vector control strategy. Chemical control is an effec-tive strategy used extensively in daily life. Synthetic insecti-cides are today at the forefront of mosquito-controlling agents.However, the environmental threat that these chemicals pose,effects on nontarget organisms, and the resistance of mosqui-toes to insecticides have all increased during the last fivedecades (Wattanachai and Tintanon 1999; Amer and
Mehlhorn 2006a, b). Biopesticides provide an alternative tosynthetic pesticides because of their generally low environ-mental pollution, low toxicity to humans, and other advan-tages (Liu et al. 2000). Many herbal products have been usedas natural insecticides before the discovery of synthetic or-ganic insecticides (ICMR Bulletin 2003). Natural products ofplant origin with insecticidal properties have been tried in therecent past in order to control a variety of insect pests andvectors. Many approaches have been developed to controlmosquito menace. One such approach to prevent mosquito-borne disease is by killing mosquito at larval stage. Thecurrent mosquito control approach is based on synthetic in-secticides. Even though they are effective, they created manyproblems like insecticide resistance (Liu et al. 2005). This hasnecessitated the need for a research and development ofenvironmentally safe, biodegradable indigenous method forvector control. Phytoextracts are emerging as potential mos-quito control agents, with low-cost, easy-to-administer, andrisk-free properties as compared to isolated or synthesizedbiopesticides and can be used successfully in mosquito man-agement (Rahuman and Venktesan 2008). Plants may be asource of alternative agents for control of vectors because theyare rich in bioactive chemicals, are active against a limitednumber of species including specific target insects, and arebiodegradable. Phytochemical insecticides have receivedmuch attention, in this regard, as they are considered to bemore environmentally biodegradable and considered saferthan synthetic insecticides (Moretti et al. 2002; Cetin et al.2004). Many researchers have reported on the effectiveness ofplant extract against mosquito larvae (Govindarajan et al.2008a; Govindarajan 2010a; Pushpanathan et al. 2006).
Mullai et al. (2008) have reported that the leaf extract ofCitrullus vulgaris with different solvents (e.g., benzene, pe-troleum ether, ethyl acetate, and methanol) was tested forlarvicidal, ovicidal, repellent, and insect growth regulatoryactivities against A. stephensi . Murugan and Jeyabalan(1999) reported that Leucas aspera , Ocimum sanctum ,Azadirachta indica , Allium sativum , and Curcuma longahad a strong larvicidal, antiemergence, adult repellency, andantireproductive activity against A. stephensi . Babu andMurugan (2000) investigated that the larvicidal effect of res-inous exudate from the tender leaves of Azadirachta indica.The leaf extract of Acalypha indica with different solvents—benzene, chloroform, ethyl acetate, and methanol—has beentested for larvicidal, ovicidal activity, and ovipositionattractancy against A. stephensi (Govindarajan et al. 2008b).Sivagnaname and Kalyanasundaram (2004) evaluated themethanolic extracts of the leaves of Atlanta monophylla(Rutaceae) for mosquitocidal activity against immature stagesof three mosquito species, C. quinquefasciatus , A. stephensi ,and A. aegypti in the laboratory. The ethanolic leaf extract ofCassia obtusifolia (Rajkumar and Jebanesan 2009) and thelarvicidal efficacy of the crude leaf extracts of Ficus
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benghalensis with three different solvents like methanol, ben-zene, and acetone were tested against the early second, third,and fourth instar larvae of C. quinquefasciatus , A. aegypti ,and A. stephensi . Among the three solvents, maximum effi-cacy was observed in methanol (Govindarajan 2010b).Samidurai et al. (2009) observed that the leaf extracts ofPemphis acidula were evaluated for larvicidal, ovicidal, andrepellent activities against C. quinquefasciatus and A.aegypti . The acetone, chloroform, ethyl acetate, hexane, andmethanol leaf extracts of Acalypha indica , Achyranthesaspera , L. aspera , Morinda tinctoria , and O. sanctum werestudied against the early fourth instar larvae of A. aegypti andC. quinquefasciatus (Bagavan et al. 2008). Larvicidal activityof crude hexane, ethyl acetate, petroleum ether, acetone, andmethanol extracts of the leaf of five species of cucurbitaceousplants, Citrullus colocynthis , Coccinia indica , Cucumissativus , Momordica charantia , and Trichosanthes anguinawas tested against the early fourth instar larvae of A. aegyptiL. and C. quinquefasciatus (Rahuman et al. 2008).
Elango et al. (2009) have reported that the leaf acetone,chloroform, ethyl acetate, hexane, and methanol extracts ofAegle marmelos , Andrographis lineata , Andrographispaniculata , Cocculus hirsutus , Eclipta prostrata , andTagetes erecta were tested against fourth instar larvae ofAnopheles subpictus and Culex tritaeniorhynchus . The etha-nol extract of Curcuma aeruginosa , Curcuma aromatica , andCurcuma xanthorrhiza was tested for repellent activityagainst Aedes togoi , Armigeres subalbatus , C.quinquefasciatus , and C. tritaeniorhynchus (Pitasawat et al.2003). Muthukrishnan and Puspalatha (2001) evaluated thelarvicidal activity of extracts from Calophyllum inophyllum(Clusiaceae), Rhinacanthus nasutus (Acanthaceae), Solanumsuratense (Solanaceae), Samadera indica (Simaroubaceae),and Myriophyllum spicatum (Haloragaceae) against A.stephensi . Several indigenous plants, viz., Ocimumbasilicum , Ocimum santum , Azadirachta indica , Lantanacamera , Vitex negundo , and Cleome viscosa , were studiedfor their larvicidal action on the field which collected fourthinstar larva of C. quinquefasciatus (Kalyanasundaram andDos 1985).Murugan et al. (2003) studied the interactive effectof botanicals (neem, pongamia) and L. aspera and B.sphaericus against the larvae of C. quinquefasciatus .Vahitha et al. (2002) studied the larvicidal efficacy ofPavonia zeylanica L. and Acacia ferruginea against C.quinquefasciatus Say. Shigeo et al. (2004) reported the larvi-cidal effect of neem against A. aegypti and chironomid larvae.Ovicidal effects of the seed extract of Atriplex canescens werereported against C. quinquefasciatus (Ouda et al. 1998) andthe larvicidal and repellent properties of essential oils fromvarious parts of four plant species Cymbopogon citratus ,Cinnamomum zeylanicum , Rosmarinus officinalis , andZingiber officinale against C. tritaeniorhynchus and A.subpictus (Govindarajan 2011). Su andMulla (1998) reported
the ovicidal activity of the neem product azadirachtin againstthe mosquitoes Culex tarsalis and C. quinquefasciatus .
Erythrina indica belonging to the family Fabaceae alsoknown as Indian coral tree or tropical coral tree or Tiger’sclow or Moochy wood tree or variegated coral tree, sun-shine tree, coral bean, and Kalyana murungai (Tamil). E.indica is a compact shrub with knobby stems. It possesdense clusters of deep crimson flowers that spread broadlyopen. E. indica is a medium-sized, spiny, deciduous treenormally growing to 6–9 m tall. Young stems and branchesare thickly armed with stout conical spines up to 8-mmlong, which fall off after 2–4 years rarely: a few spinespersist and are retained with the corky bark. Bark is smoothand green when young, exfoliating in papery flakes, be-coming thick, corky, and deeply fissured with age. Leavesare trifoliate, alternate, bright emerald green, petioles arelong about 6–15 cm, rachis 5–30-cm long, and prickly, andleaflets are smooth, shiny, and broader than long, 8–20 by5–15 cm, ovate to acuminate with an obtusely pointed end.Leaf petiole and rachis are spiny.
Vernacular names English name: Indian coral tree,Tiger’s clow, Moochy wood tree,sunshine tree
As far as our literature survey could ascertain, no infor-mation was available on the larvicidal, ovicidal, andadulticidal activities of the experimental plant species givenhere against A. stephensi , A. aegypti , and C. quinquefasciatus .Therefore, the aim of this study was to investigate the mosquitolarvicidal, ovicidal, and adulticidal activities of the differentsolvent extracts of E. indica plant species from Tamil Nadu,India. This is the first report on the mosquito larvicidal,ovicidal, and adulticidal activity of the solvent extractsof selected plant.
Materials and methods
Collection of plants
The healthy leaves of E. indica (Fig. 1) were collected fromSethiyathope, Tamil Nadu, India. It was authenticated by aplant taxonomist from the Department of Botany, Annamalai
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University, Annamalainagar. A voucher specimen was depos-ited at the Department of Zoology, Annamalai University.
Extraction
The healthy leaves were washed with sterile distilled water,shade dried, and finely ground. The finely ground leaf powder(500 g/solvent) was extracted with five different solvents, viz.,hexane, benzene, chloroform, ethyl acetate, and methanol,using Soxhlet extraction apparatus, and the extraction wascontinued till visibly no further extraction is possible (byobserving the colour of the extracted portion). The solventsfrom the extracts were removed using a rotary vacuum evap-orator to collect the crude extract and stored at 4 °C. Standardstock solutions were prepared at 1 % by dissolving the resi-dues in ethanol. From this stock solution, different concentra-tions were prepared and these solutions were used for larvi-cidal, ovicidal, and adulticidal bioassays.
Test organisms
The laboratory-bred pathogen-free strains of mosquitoes werereared in the vector control laboratory, Department ofZoology, Annamalai University. The larvae were fed on dogbiscuits and yeast powder in the 3:1 ratio. At the time of adultfeeding, these mosquitoes were 3–4 days old after emergences(maintained on raisins and water) and were starved for 12 hbefore feeding. Each time, 500 mosquitoes per cage were fedon blood using a feeding unit fitted with parafilm as
membrane for 4 h. A. aegypti feeding was done from 12 noonto 4:00 p.m. and A. stephensi and C. quinquefasciatus werefed during 6:00 to 10:00 p.m. A membrane feeder with thebottom end fitted with parafilm was placed with 2.0 ml of theblood sample (obtained from a slaughter house by collectingin a heparinized vial and stored at 4 °C) and kept over a nettedcage of mosquitoes. The blood was stirred continuously usingan automated stirring device, and a constant temperature of37 °C was maintained using a water jacket circulating system.After feeding, the fully engorged females were separated andmaintained on raisins. Mosquitoes were held at 28±2 °C, 70–85 % relative humidity, with a photoperiod of 12-h light and12-h dark.
Larvicidal bioassay
The larvicidal activity of the plant crude extracts was evalu-ated as per the method recommended by World HealthOrganization (2005). Batches of 25 third instar larvae weretransferred to small disposable paper cups, each containing200 ml of water. The appropriate volume of dilution wasadded to 200 ml water in the cups to obtain the desired targetdosage, starting with the lowest concentration (30–250 ppm).Four replicates were set up for each concentration, and anequal number of controls were set up simultaneously using tapwater. To this, 1 ml of ethanol was added. The LC50 (lethalconcentration that kills 50 % of the exposed larvae) and LC90
(lethal concentration that kills 90 % of the exposed larvae)values were calculated after 24 h by probit analysis (Finney1971).
Ovicidal activity
For ovicidal activity, slightly modified method of Su andMulla (1998) was performed. A. stephensi , A. aegypti ,and C. quinquefasciatus eggs were collected from vectorcontrol laboratory, Department of Zoology, AnnamalaiUniversity. The leaf extracts were diluted in the ethanolto achieve various concentrations ranging from 50 to300 ppm. Eggs of these mosquito species (100) wereexposed to each concentration of leaf extracts. After24 h treatment, the eggs from each concentration wereindividually transferred to distilled water cups for hatch-ing assessment after counting the eggs under a micro-scope. Each experiment was replicated six times alongwith appropriate control. The hatch rates were assessed48 h posttreatment by following the formula:
% hatchability ¼ No: of hatched larvae
Total number of eggs� 100
Fig. 1 E. indica plant
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Adulticidal bioassay
Adulticidal bioassay was performed by WHO method (WorldHealth Organization 1981). Appropriate concentrations ofcrude extracts were diluted with ethanol to achieved differentconcentrations and applied on Whatman no. 1 filter papers(size 12×15 cm). Control papers were treated with ethanolunder similar conditions. Adulticidal activity of the crudeextracts was evaluated at various concentrations (from 40 to300 ppm). Twenty female mosquitoes were collected andgently transferred into a plastic holding tube. The mosquitoeswere allowed to acclimatize in the holding tube for 1 h andthen exposed to test paper for 1 h. At the end of exposureperiod, the mosquitoes were transferred back to the holdingtube and kept 24 h for recovery period. A pad of cotton soakedwith 10 % glucose solution was placed on the mesh screen.Each experiment was replicated five times along with appro-priate control. Mortality of mosquitoes was determined at theend of 24 h recovery period. LD50 and LD90 with their 95 %confidence limits were determined using log probit analysistest (Finney 1971).
Statistical analysis
The average larval (adult) mortality data were subjected toprobit analysis for calculating LC50 (LD50), LC90 (LD90), andother statistics at 95 % confidence limits of upper confidencelimit (UCL) and lower confidence limit (LCL) values, andslope, regression equation, and chi-square test were calculatedusing the SPSS 14.0 (Statistical Package of Social SciencesInc., USA) software.
Results
The results of the larvicidal activity of crude hexane, benzene,chloroform, ethyl acetate, and methanol solvent leaf extractsof E. indica against the larvae of three important vectormosquitoes, viz., A. stephensi , A. aegypti , and C.quinquefasciatus , are presented in Tables 1, 2, 3, and 4 andFig. 2. Among the extracts tested, the highest larvicidal activ-ity was observed in methanol extract against A. stephensifollowed by A. aegypti and C. quinquefasciatus with theLC50 and LC90 values of 69.43, 75.13 and 91.41 ppm and125.49, 134.31, and 167.14 ppm, respectively. The 95 %confidence limits LC50 (LCL–UCL) and LC90 (LCL–UCL),regression equation, slope, and chi-square values were alsocalculated. The mean percent egg hatchabilities of A.stephensi , A. aegypti , and C. quinquefasciatus were testedwith five different solvents at different concentrations of E.indica leaf extracts, and the results are listed in Table 5. Thepercent hatchability was inversely proportional to the concen-tration of extract and directly proportional to the eggs. Among
the extracts tested for ovicidal activity against A. stephensi , A.aegypti , and C. quinquefasciatus , the methanol extract ofAsparagus racemosus exerted 100 % mortality (zero hatch-ability) at 150, 200, and 250 ppm, respectively. Control eggsshowed above 99.3–100 % hatchability. The results of theadulticidal activity of hexane, benzene, chloroform, ethylacetate, and methanol solvent leaf extracts of E. indica againstthe adult of three important vector mosquitoes, viz., A.stephensi , A. aegypti , and C. quinquefasciatus , are presentedin Tables 6, 7, 8, and 9 and Fig. 3. The plant crude extractsshowed dose-dependent mortality. At higher concentrations,the adult showed restless movement for some times withabnormal wagging and then died. Among the extracts tested,the highest adulticidal activity was observed in methanolextract against A. stephensi followed by A. aegypti and C.quinquefasciatus. The LD50 and LD90 values of E. indica leafextracts against adulticidal activity of (hexane, benzene, chlo-roform, ethyl acetate, and methanol) A. stephensi , A. aegypti ,and C. quinquefasciatus were the following: A. stephensi ,LD50 values were 88.76, 121.53, 113.74, 128.92, and140.79 ppm, and LD90 values were 160.83, 222.78, 208.14,233.22, and 255.10 ppm; A. aegypti LD50 values were 94.09,126.72, 121.91, 134.24, and 149.75 ppm, and LD90 valueswere 169.01, 229.30, 219.88, 241.14, and 271.73 ppm; andC.quinquefasciatus LD50 values were 119.64, 129.82, 127.70,137.97, and 153.75 ppm, and LD90 values were 219.77,240.26, 229.08, 253.09, and 280.09 ppm, respectively. Nomortality was recorded in the control. The 95 % confidencelimits LD50 (LCL–UCL) and LD90 (LCL–UCL), slope, re-gression equation, and chi-square test were also calculated.
Discussion
Phytochemicals may serve as suitable alternatives to syntheticinsecticides in the future as these are relatively safe, inexpen-sive, and are readily available in many areas of the world.Different parts of plants contain a complex of chemicals withunique biological activity which is thought to be due to toxinsand secondary metabolites, which act asmosquitocidal agents.Furthermore, the crude extracts may be more effective com-pared to the individual active compounds, due to naturalsynergism that discourages the development of resistance inthe vectors. Our results showed that the crude hexane, ben-zene, chloroform, ethyl acetate, and methanol solvent extractsof E. indica were effective against the eggs, larvae, and adultof three important vector mosquitoes, viz., A. stephensi , A.aegypti , and C. quinquefasciatus. This result is also compa-rable to earlier reports of Singh et al. (2003) who observed thelarvicidal activity of Ocimum canum oil against vector mos-quitoes, namely, A. aegypti and C. quinquefasciatus(LC50=301 ppm) and A. stephensi (234 ppm). Traboulsiet al. (2005) reported the larvicidal activity of essential oils
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of Citrus sinensis , Eucalyptus spp., Ferula hermonis , Laurusnobilis , and Pinus pinea against Culex pipiens . LC50 valueswere 60.0, 120.0, 44.0, 117.0, and 75.0 ppm, respectively. Theessential oil of Zingiber officinalis exhibited more effectivelarvicides than the reported plants. The essential oil of Tagetes
minuta , providing a repellency of 90 % protection for 2 h wasobserved by Tyagi et al. (1994).
Earlier authors reported that the petroleum ether extract ofR. nasutus possessed larvicidal effects with LC50 values be-tween 3.9 and 11.5 mg/l, and Derris elliptica showed LC50
Table 1 Percentage mortality of mosquito larvae of Culex quinquefasciatus, Aedes aegypti , and Anopheles stephensi exposed to different concentra-tions of different solvent leaf crude extracts of E. indica
Solvents Culex quinquefasciatus Aedes aegypti Anopheles stephensi
Concentration (ppm) % of mortality ± SDa Concentration (ppm) % of mortality ± SDa Concentration (ppm) % of mortality ± SDa
Hexane Control50100150200250
0±0.019±0.940±1.461±0.975±0.994±1.3
Control50100150200250
0±0.026±1.344±0.865±0.978±1.397±0.9
Control50100150200250
0±0.028±0.847±0.966±1.381±0.999±0.5
Benzene Control50100150200250
0±0.026±1.343±0.965±0.977±0.996±0.8
Control4080120160200
0±0.019±0.940±0.858±1.373±1.392±0.8
Control4080120160200
0±0.021±0.943±0.961±0.975±1.594±1.3
Chloroform Control4080120160200
0±0.022±1.343±0.962±1.378±1.395±0.9
Control4080120160200
0±0.024±1.446±1.365±0.980±1.497±0.9
Control4080120160200
0±0.027±0.949±0.968±0.883±0.999±0.5
Ethyl acetate Control50100150200250
0±0.029±1.547±0.968±1.481±0.999±0.5
Control4080120160200
0±0.022±1.343±0.962±1.377±0.995±0.9
Control4080120160200
0±0.025±0.946±1.365±1.579±1.596±0.8
Methanol Control4080120160200
0±0.027±0.948±0.867±0.981±0.999±0.5
Control306090120150
0±0.021±1.545±0.961±0.978±1.396±0.9
Control306090120150
0±0.026±1.347±1.565±0.982±1.399±0.5
SD standard deviationa Values are mean ± SD of four replicates
Table 2 LC50, LC90, slope, regression equation, and chi-square analysis of larvicidal activity of different solvent leaf extracts ofErythrina indica againstCulex quinquefasciatus
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at P<0.05
782 Parasitol Res (2014) 113:777–791
values between 11.2 and 18.84 mg/l against A. aegypti , C.quinquefasciatus , Anopheles dirus , and Mansonia uniformis(Komalamisra et al. 2005). The same extracts of Argemonemexicana , Jatropha curcas , Parapsyche extensa , andWithania somnifera showed acute toxicity causing 100 %mortality at 1,000, 500, and 250 ppm, respectively, againstC. quinquefasciatus larvae (Karmegam et al. 1997). Solanumxanthocarpum fruit petroleum ether extract was observed asthe most toxic with LC50 values of 62.62 ppm after 24 h and59.45 ppm after 48 h of exposure period against the larvae ofC. quinquefasciatus (Mohan et al. 2005). Mathew et al.(2009) reported that leaf chloroform extracts of Nyctanthesarbortristis showed lethal values (LC50 of 0526.3 and780.6 ppm (24 h) and LC50 of 0303.2 and 518.2 ppm(48 h)) against A. aegypti and A. stephensi , respectively.Flower methanol extracts of the above plants showed lethalvalues (LC50=679.4 and 244.4 ppm; LC90=1,071.3 and433.7 ppm) against A. stephensi after 24 and 48 h, respective-ly. Clitoria ternatea leaf methanol extract showed dose-dependent larvicidal activity against A. stephensi with LC50
values of 555.6 ppm (24 h) and 867.3 ppm (48 h), also the rootextracts with LC50 value of 340 ppm (48 h). Seed extractshowed larvicidal activity (LC50=0116.8 and 195 ppm) after24 h and (LC50=065.2 and 154.5 ppm) after 48 h treatmentagainst A. stephensi and A. aegypti , respectively. Larvicidalactivity of flowermethanol extract showed LC50 values of 233and 302.5 ppm against A. stephensi and A. aegypti ,
respectively, after 48 h treatment. Methanol extract showedthe lowest LD values against several instars of larvae and 50adult (121.59, 142.73, 146.84, 202.98, 290.65, 358.42, and300.03 μg/cm2, respectively) which indicates the highest tox-icity or insecticidal activity (Ashraful Alam et al. 2009).Sharma et al. (2005) reported that the acetone extract ofNerium indicum and Thuja orientelis has been studied withLC50 values of 200.87, 127.53, 209.00, and 155.97 ppmagainst III instar larvae of A. stephensi and C.quinquefasciatus , respectively.
Khanna et al. (2011) have reported that the larvicidal crudeleaf extract ofGymnema sylvestre showed the highest mortal-ity in the concentration of 1,000 ppm against the larvae of A.subpictus (LC50=166.28 ppm) and against the larvae of C.quinquefasciatus (LC50=186.55 ppm), and the maximumefficacy was observed in gymnemagenol compound isolatedfrom petroleum ether leaf extract of G. sylvestre with LC50
values against the larvae of A. subpictus at 22.99 ppm andagainst C. quinquefasciatus at 15.92 ppm. Prophiro et al.(2012) reported that the susceptibility of larvae was deter-mined under three different temperatures, 15, 20, and 30 °C,with lethal concentrations for Copaifera sp. ranging fromLC50 of 47 mg/L to LC90 of 91 mg/L and for Carapaguianensis LC50 of 136 to LC90 of 551 mg/L.Santhoshkumar et al. (2011) reported that the maximum effi-cacy was observed in crude methanol and aqueous leaf ex-tracts of Nelumbo nucifera against the larvae of A. subpictus
Table 3 LC50, LC90, slope, regression equation, and chi-square analysis of larvicidal activity of different solvent leaf extracts ofErythrina indica againstAedes aegypti
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at P<0.05
Parasitol Res (2014) 113:777–791 783
(LC50=08.89 and 11.82 ppm and LC90=028.65 and36.06 ppm, respectively) and against the larvae of C.quinquefasciatus (LC50=09.51 and 13.65 ppm and LC90=028.13 and 35.83 ppm, respectively). In the present resultswith Calotropis gigantea against C. quinquefasciatus , theLC50 value of I instar was 104.660 %, II instar was
127.71 ppm, III instar was 173.75 ppm, and IV instarwas 251.65 ppm. The LC90 value of I instar was268.67 ppm, II instar was 323.50 ppm, III instar was432.11 ppm, and IV instar was 581.66 ppm. The LC50
value of pupae was 314.70 ppm, and the LC90 value ofpupae was 665.04 ppm.
LC50
LC90
LC90
LC50
LC50
LC90
Fig. 2 a–c Graph showing theLC50 and LC90 values of threeimportant mosquito larvae
784 Parasitol Res (2014) 113:777–791
Kovendan et al. (2012) have reported hexane, chloroform,ethyl acetate, and methanol extract of J. curcas with LC50
values of 230.32, 212.85, 192.07, and 113.23 ppm; Hyptissuaveolens with LC50 values of 213.09, 217.64, 167.59, and86.93 ppm; Abutilon indicum with LC50 values of 204.18,155.53, 166.32, and 111.58 ppm; and L. aspera with LC50
values of 152.18, 118.29, 111.43, and 107.73 ppm, respec-tively, against third instar larvae of C. quinquefasciatus .Mahesh Kumar et al. (2012) have reported that 43%mortalitywas noted at first instar larvae by the treatment of S.xanthocarpum at 50 ppm, whereas it has been increased to92 % at 650 ppm; 21.2 % mortality was noted at 50 ppm of S.xanthocarpum leaf extract treatment at 24 h exposure. TheLC50 values of first to fourth instar larvae and pupae were155.29, 198.32, 271.12, 377.44, and 448.41 ppm, respective-ly. The LC90 values of first to fourth instar larvae and pupaewere 687.14, 913.10, 1,011.89, 1,058.85, and 1,141.65 ppm,respectively. Bansal et al. (2009a, b) have reported methanolicextracts from fruits without seeds, and S. xanthocarpum wasevaluated against larvae of Anopheles culicifacies , A.stephensi , A. aegypti , and C. quinquefasciatus , the importantvector mosquitoes. The LC50 values were 79.6, 91.7, and131.7; 131.4, 186.9, and 195.6; 273.4, 290.9, and 377.6; and384.9, 450.6, and 520.0 mg l−1 against mosquito vectors.
Thirteen oils from 41 plants (camphor, thyme, amyris,lemon, cedar wood, frankincense, dill, myrtle, juniper, blackpepper, verbena, helichrysum, and sandalwood) induced100 % mortality after 24 h or even after shorter periods. The
pest oils were tested against third instar larvae of the threemosquito species in concentrations of 1, 10, 50, 100, and500 ppm. The lethal concentration 50 values of three oilsranged between 1 and 101.3 ppm against A. aegypti , between9.7 and 101.4 ppm for A. stephensi , and between 1 and50.2 ppm for C. quinquefasciatus (Amer and Mehlhorn2006b). The LC50 and LC90 values ofCassia tora leaf extractsagainst adulticidal activity of hexane, chloroform benzene,acetone, and methanol (C. quinquefasciatus , A. aegypti , andA. stephensi ) were the following: for C. quinquefasciatus ,LC50 values were 338.81, 315.73, 296.13, 279.23, and261.03 ppm and LC90 values were 575.77, 539.31, 513.99,497.06, and 476.03 ppm; for A. aegypti , LC50 values were329.82, 307.3, and 252.03 ppm and LC90 values were 563.24,528.33, 496.92, 477.61, and 448.05 ppm; and for A.stephensi , LC50 values were 317.28, 300.30, 277.51,263.35, and 251.43 ppm and LC90 values were 538.22,512.90, 483.78, 461.08, and 430.70 ppm, respectively(Amerasan et al. 2012).
Earlier authors reported that the methanol leaf extracts of V.negundo , Vitex trifolia , Vitex peduncularis , and Vitexaltissima were used for larvicidal assay with LC50 values of212.57, 41.41, 76.28, and 128.04 ppm, respectively, againstthe early fourth instar larvae of C. quinquefasciatus(Kannathasan et al. 2007). The same extracts of Euphorbiatirucalli latex and stem bark were evaluated for larvicidalactivity against laboratory-reared larvae of C.quinquefasciatus with LC50 values of 177.14 and
Table 5 Ovicidal activity of different solvent leaf extracts of Erythrina indica against Anopheles stephensi , Culex quinquefasciatus, and Aedes aegypti
513.387 mg/l, respectively (Yadav et al. 2002). Larvicidalactivity of the same extracts of Murraya koenigii ,Coriandrum sativum , Ferula asafoetida , and T. foenumgraceum were tested out using different concentrations ofeach plant (range 25–900 ppm) against A. aegypti larvae
(Harve and Kamath 2004). The compound like diterpenoidfurans 6alpha-hydroxyvouacapan-7beta, 17beta-lactone (1),6alpha,7beta-dihydroxyvouacapan-17beta-oic acid (2), andmethyl6alpha,7beta-dihydroxyvouacapan-17beta-oate (3)from seeds of Pterodon spolygalaeflorus exhibited LC50
Table 6 Percentage mortality of mosquito adult ofCulex quinquefasciatus , Aedes aegypti , andAnopheles stephensi exposed to different concentrationsof different solvent leaf crude extracts of Erythrina indica
Solvents Culex quinquefasciatus Aedes aegypti Anopheles stephensi
Concentration (ppm) % of mortality ± SDa Concentration (ppm) % of mortality ± SDa Concentration (ppm) % of mortality ± SDa
Hexane Control60120180240300
0±0.022±1.143±1.161±0.878±1.192±1.1
Control60120180240300
0±0.023±1.144±0.861±1.380±1.094±0.8
Control60120180240300
0±0.025±0.748±1.663±1.182±0.598±0.5
Benzene Control60120180240300
0±0.024±0.846±0.875±1.282±1.395±0.7
Control50100150200250
0±0.022±1.137±0.559±0.874±0.892±1.1
Control50100150200250
0±0.023±0.540±1.062±0.575±0.794±0.8
Chloroform Control50100150200250
0±0.023±0.540±1.061±0.877±0.595±0.7
Control50100150200250
0±0.025±0.742±1.164±0.879±0.897±0.5
Control50100150200250
0±0.028±1.146±0.869±1.381±0.899±0.4
Ethyl acetate Control60120180240300
0±0.028±0.548±1.177±1.185±1.097±0.5
Control50100150200250
0±0.024±0.840±0.763±0.876±1.595±1.2
Control50100150200250
0±0.026±0.843±0.565±0.278±0.596±0.8
Methanol Control50100150200250
0±0.028±0.545±1.061±0.879±0.898±0.5
Control4080120160200
0±0.026±0.845±0.763±1.182±1.199±0.4
Control4080120160200
0±0.029±0.848±0.865±1.086±0.8100±0.0
SD standard deviationa Values are mean ± SD of four replicates
Table 7 LD50, LD90, slope, regression equation, and chi-square analysis of adulticidal activity of different solvent leaf extracts of Erythrina indicaagainst Culex quinquefasciatus
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at P<0.05
786 Parasitol Res (2014) 113:777–791
values of 50.08, 14.69, and 21.76 μg/ml against fourth instarA. aegypti larvae, respectively (Omena et al. 2006). Siddiquiet al. (2004) have reported that the compounds spipnoohine(1) and pipyahyine (2) isolated from the petroleum etherextract of dried ground whole fruits of Piper nigrum exhibitedtoxicity at 35.0 and 30.0 ppm, respectively, against fourthinstar larvae of A. aegypti . This has been observed earlier:fraction A1 of ethanol from Sterculia guttata seed extract wasfound to be most promising; its LC50 was 21.552 and35.520 ppm against C. quinquefasciatus and A. aegypti ,respectively (Katade et al. 2006).
Larvicidal efficacies of methanol extracts ofM. charantia ,T. anguina , Luffa acutangula , Benincasa cerifera , and C.vulgaris tested with LC50 values were 465.85, 567.81,839.81, 1,189.30 and 1,636.04 ppm, respectively, against thelate third larval age group of C. quinquefasciatus (Prabakarand Jebanesan 2004). The aqueous extract of R. nasutusshowed LC50 values of 5,124 and 9,681 mg/l against C.quinquefasciatus and A. Aegypti , respectively (Chansanget al. 2005). Of the ethanol extracts of the aerial parts fromfive Labiatae species, Teucrium divaricatum was the mosttoxic, followed by Mentha longifolia , Melissa officinalis ,Salvia sclarea , and Mentha pulegium against the third andfourth instar larvae of C. pipiens with LC50 values of 18.6,26.8, 39.1, 62.7, and 81.0 ppm, respectively (Cetin et al.2006). Extract from Lavandula stoechas (Labiatae) showedLC50 values of 89 mg l−1 against fourth instar larvae of
C. pipiens molestus (Traboulsi et al. 2002). The crude meth-anolic extract of Trichilia americana exhibited strongantifeedant activity in a choice leaf disc bioassay with0.18 μg cm−2 extract deterring feeding by 50 % (Wheelerand Isman 2001), and the crude seed extracts of Origanumvulgare showed antifeedant activity against third instar larvaeof Trichoplusia ni (Akhtar and Isman 2004). Alkaloid extractof the plant has been found to deter feeding of S. litura larvaeat 0.01 % (Verma et al. 1986). Reduced weight gain comparedto control was also observed after 72 h on S. litura larvaetreated with meliatoxin A2 and meliatoxin B1, both com-pounds isolated from Melia azedarach at 480 and600 μg cm−2, respectively (Macleod et al. 1990).
Earlier authors reported that the isolated compoundneemarin from Azadirachta indica exhibited LC50 and LC90
values of 0.35 and 1.81 mg/l for A. stephensi and 0.69 and3.18 mg/l for C. quinquefasciatus (Vatandoost and Vaziri2004); leptostachyol acetate compound isolated from the rootsof Phryma leptostachya with LC50 values of 0.41, 2.1, and2.3 ppm against third instar larvae of C. pipiens pallens , A.aegypti , and Ochlerotatus togoi (Park et al. 2005);vilasininoid and two havanensinoids isolated from the chloro-form fractions of the methanol extracts of the root barks ofTurraea wakefieldii and Turraea floribunda showed LD50
values of 7.1, 4.0, and 3.6 ppm respectively, against thirdinstar larvae of Anopheles gambiae (Ndung’u et al. 2004).Shaalan et al. (2006) have reported that the LC50 value of
Table 8 LD50, LD90, slope, regression equation, and chi-square analysis of adulticidal activity of different solvent leaf extracts of Erythrina indicaagainst Aedes aegypti
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at P<0.05
Table 9 LD50, LD90, slope, regression equation, and chi-square analysis of adulticidal activity of different solvent leaf extracts of Erythrina indicaagainst Anopheles stephensi
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square; df degrees of freedoma Significant at P<0.05
Parasitol Res (2014) 113:777–791 787
acetone extracts of Khaya saenegalensis and Daucus carotawere 20.12 and 236.00 mg/l, respectively, against fourth in-stars of Culex annulirostris . Rahuman and Venktesan (2008)reported the petroleum ether extract of C. colocynthis , meth-anol extracts of Cannabis indica , Cannabis sativus , and M.charantia , and acetone extract of T. anguina against thelarvae of A. aegypti (LC50=74.57, 309.46, 492.73,199.14, and 554.20 ppm) and against C. quinquefasciatus(LC50=88.24, 377.69, 623.80, 207.61, and 842.34 ppm), re-spectively. Similarly, a piperidine alkaloid, pipernonaline, iso-lated from the fruit methanol extract of Piper longum showedLD50 value of 0.21 mg/l C. pipiens pallens larvae (Lee 2000).
A new tetranortriterpenoid, meliatetraolenone [24,25,26,27-tetranorapotirucalla-(apoeupha)-6alpha-O-methyl, 7alpha-senecioyl (7-deacetyl)-11alpha, 12alpha, 21,23-tetrahydroxy-21, 23- epoxy-2, 14,20(22)-trien-1, 16-dione] (1), was isolatedfrom the methanolic extract of fresh leaves of Azadirachtaindica along with the known compound odoratone (3) whichshowed mortality on fourth instar larvae of mosquitoes (A.stephensi) with LC50 values of 16 and 154 ppm, respectively(Siddiqui et al. 2003); two new triterpenoids, 22,23-dihydronimocinol (1) and desfurano-6alpha-hydroxyazadiradione (2), were isolated from a methanolicextract of the fresh leaves of Azadirachta indica along with
LD50
LD90
LD50
LD90
LD50
LD90
a
b
c
Fig. 3 a–c Graph showing theLD50 and LD90 values of threeimportant adult mosquitoes
788 Parasitol Res (2014) 113:777–791
a known meliacin; 7alpha-senecioyl-(7-deacetyl)-23-O -methylnimocinolide showed mortality for fourth instar larvaeof the mosquito (A. stephensi ), with LC50 values of 60 and43 ppm, respectively (Siddiqui et al. 2002).
In conclusion, our findings showed that the plant E.indica exhibits larvicidal, ovicidal, and adulticidal activ-ity against three important vector mosquitoes. Theseresults could encourage the search for new active natu-ral compounds offering an alternative to synthetic insec-ticides from other medicinal plant. E. indica extractsmay contribute greatly to save the environment and toan overall reduction in the population density of threesignificant vectors (A. stephensi , A. aegypti , and C.quinquefasciatus ). Also, our results open the possibilityfor further investigations of the efficacy of larvicidal,ovicidal, and adulticidal properties of natural productextracts.
Acknowledgments The authors are grateful to the Indian Council ofMedical Research (ICMR ref. letter no. 5/8-7(246)/2012-ECD-II), NewDelhi, India for providing financial assistance and would like to thankDr. N. Indra, Professor and Head of the Department of Zoology,Annamalai University for the laboratory facilities provided. The authorswould also like to acknowledge the cooperation of staff members of theVCRC (ICMR), Pondicherry.
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