ORIGINAL PAPER Ovicidal, larvicidal and adulticidal properties of Asparagus racemosus (Willd.) (Family: Asparagaceae) root extracts against filariasis (Culex quinquefasciatus ), dengue (Aedes aegypti ) and malaria (Anopheles stephensi ) vector mosquitoes (Diptera: Culicidae) Marimuthu Govindarajan & Rajamohan Sivakumar Received: 21 December 2013 /Accepted: 10 January 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Several diseases are associated to the mosquito–hu- man interaction. Mosquitoes are the carriers of severe and well- known illnesses such as malaria, arboviral encephalitis, dengue fever, chikungunya fever, West Nile virus and yellow fever. These diseases produce significant morbidity and mortality in humans and livestock around the world. The present investiga- tion was undertaken to study the ovicidal, larvicidal and adulticidal activities of crude hexane, ethyl acetate, benzene, chloroform and methanol extracts of root of Asparagus racemosus were assayed for their toxicity against three important vector mosquitoes, viz., Culex quinquefasciatus , Aedes aegypti and Anopheles stephensi (Diptera: Culicidae). The mean percent hatchability of the eggs was observed after 48 h post-treatment. The percent hatchability was inversely proportional to the con- centration 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 Asparagus racemosus against Culex quinquefasciatus , Aedes aegypti and Anopheles stephensi exerted 100 % mortality (zero hatchability) at 375, 300 and 225 ppm, respectively. Control eggs showed 99–100 % hatchability. The larval mortality was observed after 24 h of exposure. All extracts showed moderate larvicidal effects; however, the highest larval mortality was found in methanol extract of root of Asparagus racemosus against the larvae of Culex quinquefasciatus , Aedes aegypti and Anopheles stephensi with the LC 50 and LC 90 values were 115.13, 97.71 and 90.97 ppm and 210.96, 179.92, and 168.82 ppm, respectively. 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 Anopheles stephensi followed by Aedes aegypti and Culex quinquefasciatus with the LD 50 and LD 90 values were 120.44, 135.60, and 157.71 ppm and 214.65, 248.35, and 290.95 ppm, respectively. No mortality was recorded in the control. The finding of the present investigation revealed that the root extract of Asparagus racemosus possess remarkable ovicidal, larvicidal and adulticidal activity against medically important vector mosquitoes and this is the low cost and ideal eco-friendly approach for the control of mosquitoes. This is the first report on the mosquito ovicidal, larvicidal and adulticidal activities of the reported Asparagus racemosus root. Introduction Mosquito-borne diseases have an economic impact, including loss in commercial and labour outputs, particularly in coun- tries with tropical and subtropical climates. However, no part of the world is free from vector-borne diseases (Fradin and Day 2002). Mosquitoes transmit a variety of diseases, such as yellow fever, dengue fever, malaria, several forms of enceph- alitis and filariasis. Aedes aegypti is generally known as a vector for an arbovirus responsible for dengue fever, which is endemic to Southeast Asia, the Pacific island area, Africa and the Americas. This mosquito is also the vector of yellow fever in Central and South America and West Africa. Dengue fever has become an important public health problem as the number of reported cases continues to increase, especially 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 DOI 10.1007/s00436-014-3784-1
Received: 21 December 2013 /Accepted: 10 January 2014# Springer-Verlag Berlin Heidelberg 2014
Abstract Several diseases are associated to the mosquito–hu-man interaction. Mosquitoes are the carriers of severe and well-known illnesses such as malaria, arboviral encephalitis, denguefever, chikungunya fever, West Nile virus and yellow fever.These diseases produce significant morbidity and mortality inhumans and livestock around the world. The present investiga-tion was undertaken to study the ovicidal, larvicidal andadulticidal activities of crude hexane, ethyl acetate, benzene,chloroform and methanol extracts of root of Asparagusracemosuswere assayed for their toxicity against three importantvector mosquitoes, viz., Culex quinquefasciatus, Aedes aegyptiand Anopheles stephensi (Diptera: Culicidae). The mean percenthatchability of the eggs was observed after 48 h post-treatment.The percent hatchability was inversely proportional to the con-centration of extract and directly proportional to the eggs. All thefive solvent extracts showedmoderate ovicidal activity; however,the methanol extract showed the highest ovicidal activity. Themethanol extract of Asparagus racemosus against Culexquinquefasciatus, Aedes aegyptiand Anopheles stephensiexerted100 % mortality (zero hatchability) at 375, 300 and 225 ppm,respectively. Control eggs showed 99–100 % hatchability. Thelarval mortality was observed after 24 h of exposure. All extractsshowed moderate larvicidal effects; however, the highest larvalmortality was found in methanol extract of root of Asparagusracemosus against the larvae of Culex quinquefasciatus, Aedesaegypti and Anopheles stephensiwith the LC50 and LC90 valueswere 115.13, 97.71 and 90.97 ppm and 210.96, 179.92, and168.82 ppm, respectively. The adult mortality was observed after
24 h recovery period. The plant crude extracts showed dose-dependent mortality. At higher concentrations, the adult showedrestless movement for some times with abnormal wagging andthen died. Among the extracts tested, the highest adulticidalactivity was observed in methanol extract against Anophelesstephensi followed by Aedes aegypti and Culex quinquefasciatuswith the LD50 and LD90 values were 120.44, 135.60, and157.71 ppm and 214.65, 248.35, and 290.95 ppm, respectively.No mortality was recorded in the control. The finding of thepresent investigation revealed that the root extract of Asparagusracemosuspossess remarkable ovicidal, larvicidal and adulticidalactivity againstmedically important vectormosquitoes and this isthe low cost and ideal eco-friendly approach for the control ofmosquitoes. This is the first report on the mosquito ovicidal,larvicidal and adulticidal activities of the reported Asparagusracemosus root.
Introduction
Mosquito-borne diseases have an economic impact, includingloss in commercial and labour outputs, particularly in coun-tries with tropical and subtropical climates. However, no partof the world is free from vector-borne diseases (Fradin andDay 2002). Mosquitoes transmit a variety of diseases, such asyellow fever, dengue fever, malaria, several forms of enceph-alitis and filariasis. Aedes aegypti is generally known as avector for an arbovirus responsible for dengue fever, whichis endemic to Southeast Asia, the Pacific island area, Africaand the Americas. This mosquito is also the vector of yellowfever in Central and South America and West Africa. Denguefever has become an important public health problem as thenumber of reported cases continues to increase, especially
M. Govindarajan (*) :R. SivakumarUnit of Vector Biology and Phytochemistry, Department of Zoology,Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, Indiae-mail: [email protected]
Parasitol ResDOI 10.1007/s00436-014-3784-1
with more severe forms of the disease, dengue hemorrhagicfever, and dengue shock syndrome, or with unusual manifes-tations such as central nervous system involvement. Dengue isprevalent in more than 100 countries and threatens the healthof approximately 2.5 billion people. Around 80million peopleare infected annually at an attack rate of 4 % worldwide(Pancharoen et al. 2002). Culex quinquefasciatus (Say.) actsas a vector for filariasis in India. Human filariasis is a majorpublic health hazard and remains a challenging socio-economic problem in many of the tropical countries.Lymphatic filariasis caused byWuchereria bancroftiand trans-mitted by mosquito Culex quinquefasciatus is found to bemore endemic in the Indian subcontinent. It is reported thatCulex quinquefasciatus infects more than 100 million individ-uals worldwide annually (Rajasekariah et al. 1991).Anophelesstephensi Liston is the primary vector of malaria in India andother West Asian countries, Malaria remains one of the mostprevalent diseases in the tropical world. With 200 million to450 million infections annually worldwide, it causes up to 2.7million deaths. In India, malaria is one of the most importantcauses of direct or indirect infant, child and adult mortality.About 2 million confirmed malaria cases and 1,000 deaths arereported annually, although 15 million cases and 20,000deaths are estimated by WHO South East Asia RegionalOffice. India contributes 77 % of the total malaria inSoutheast Asia (WHO 2010).
The use of conventional pesticides in the water sources,however, introduces many risks to people and the environ-ment. Natural products of plant origin with insecticidal prop-erties have been tried in the recent past control of variety ofinsect pests and vectors. Mosquitoes in the larval stage areattractive targets for pesticides because mosquitoes breed inwater, and thus, it is easy to deal with them in this habitat.Many researchers have reported on the effectiveness of plantextract against mosquito larvae (Kalyanasundaram and Das1985; Prabhu et al. 2011; Kovendan et al. 2011). In recentyears, many studies on plant extracts against mosquito larvaehave been conducted around the world. The crude hexaneextract obtained from the leaf of Leucas aspera was testedfor the larvicidal activity against Culex quinquefasciatus andAedes aegypti (Maheswaran et al. 2008). Govindarajan (2009)reported that the leaf methanol, benzene and acetone extractsof Cassia fistula were studied for the larvicidal, ovicidal andrepellent activities against Aedes aegypti. The leaf extract ofAcalypha indica with different solvents, viz., benzene, chlo-roform, ethyl acetate and methanol, were tested for larvicidal,ovicidal activity and oviposition attractancy againstAnopheles stephensi (Govindarajan et al. 2008). Mullai et al.(2008) have reported that the leaf extract of Citrullus vulgariswith different solvents viz. benzene, petroleum ether, ethylacetate and methanol were tested for larvicidal, ovicidal, re-pellent and insect growth regulatory activities againstAnopheles stephensi. Elango et al. (2009) have reported that
the leaf acetone, chloroform, ethyl acetate, hexane and meth-anol extracts of Aegle marmelos, Andrographis lineata,Andrographis paniculata,Cocculus hirsutus, Eclipta prostrateand Tagetes erecta were tested against fourth-instar larvae ofAnopheles subpictus and Culex tritaeniorhynchus.Andrographis paniculata is a traditional medicinal plant thathas been used for pest control (Kuppusamy and Murugan2006).
Govindarajan and Karupannan (2011) investigated the lar-vicidal and ovicidal activities of benzene, hexane, ethyl acetate,methanol and chloroform leaf extract of Eclipta alba againstdengue vector, Aedes aegypti. Murugan et al. (2012) evaluatedthe larvicidal, pupicidal, repellent and adulticidal activity ofCitrus sinensisorange peel extract against Anopheles stephensi,Aedes aegypti and Culex quinquefasciatus. The larvicidal ac-tivity of crude petroleum ether, ethyl acetate and methanolextracts of the whole plants of Phryma leptostachya wasassayed for its toxicity against the early fourth-instar larvae ofCulex pipiens pallens (Xiao et al. 2012). Kovendan et al. (2011)investigated the larvicidal activity of methanol solvent extractsfrom leaves of Jatropha curcas and Bacillus thuringiensis var.israelensis against the lymphatic filarial vector Culexquinquefasciatus. Manimegalai et al. (2011) reported the eval-uation of larvicidal activity of the leaf and seed extracts ofAbrus precatorius against forth-instar larvae of Culexquinquefasciatus. Ethanolic, acetone and petroleum ether ex-tracts of leaves from the Egyptian plantCupressus sempervirens(Cupressaceae) were tested against third-instar larvae of themosquito Culex pipiens. The obtained results indicated thatpetroleum ether extracts were more efficient than ethanolicand acetone extracts (EL-Sheikh et al. 2011). The efficiencyof wood vinegar and extracts from three of the medicinal plantssuch as neem seed (Azadirachta indica), citronella grass(Cymbopogon nardus) and yam bean seed Pachyrhizus erosuswere tested on the third-instar larvae of mosquito (Culexquinquefasciatus) (Pangnakorn et al. 2011).
Hafeez et al. (2010) reported to evaluate the adulticidalaction of ten citrus oils against Aedes albopictus throughexposure tube method. Ovicidal and repellent activities ofmethanol leaf extract of Ervatamia coronaria and Caesalpiniapulcherrima were evaluated against Culex quinquefasciatus,Aedes aegypti and Anopheles stephensi (Govindarajan et al.2011). The larvicidal and ovicidal efficacy of different extractsof Cardiospermum halicacabum against Culex quinquefasciatusand Aedes aegypti was determined (Govindarajan 2011a). Thelarvicidal and ovicidal activity of crude hexane, ethyl acetate,benzene, chloroform and methanol extracts of the leaf of threeplants, E. alba, Cardiospermum halicacabum and Andrographispaniculata, was tested against the early third-instar larvae ofAnopheles stephensi (Govindarajan 2011b). Zahir et al. (2010)reported that the crude hexane, chloroform, ethyl acetate, acetoneand methanol extracts of Anisomeles malabarica exhibitedacaricidal and insecticidal activities against the adult of
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Haemaphysalis bispinosa and Hippobosca maculate. Theadulticidal and repellent activities of crude hexane, ethylacetate, benzene, chloroform and methanol extracts ofleaf of E. alba and Andrographis paniculata wereassayed for their toxicity against two important vectormosquitoes, viz., Culex quinquefasciatus and Aedesaegypti (Govindarajan and Sivakumar 2012a). Rohaniet al. (1997) have reported the efficacy of fewMalaysian essential oils such as Litsea elliptica,Polygonum minus and Piper aduncum as potential mos-quito adulticides, while Sulaiman et al. (2001) havereported that the essential oils of Melaleuca cajuputiand Cymbopogon nardus have adulticidal effects onAedes mosquito at high-rise flats in Kuala Lumpur.
Amerasan et al. (2012) have reported the adulticidal andrepellent activities of crude hexane, chloroform, benzene, ace-tone and methanol extracts of the leaf of Cassia tora wereassayed for their toxicity against three important vector mos-quitoes, viz., Culex quinquefasciatus, Aedes aegypti andAnopheles stephensi. The adulticidal, repellent and ovicidalpotential of the crude hexane, ethyl acetate, benzene, aqueousand methanol solvent extracts from the medicinal plantsAndrographis paniculata, Cassia occidentalis and Euphorbiahirta against the medically important mosquito vector,Anopheles stephensi, were studied (Panneerselvam andMurugan 2013). The adulticidal activity of hexane, ethyl ace-tate, benzene, chloroform and methanol leaf extracts ofCardiospermum halicacabum against Culex quinquefasciatus,Aedes aegypti and Anopheles stephensi was also determined(Govindarajan and Sivakumar 2012b). Hafeez et al. (2010)reported that the adulticidal action of ten citrus oils againstAedes albopictus through exposure tube method. Zaridahet al. (2001) have reported that the aqueous extract from thedried leaves of Andrographis paniculata showed the strongestactivity against adult worms of Brugia malayi. The adult emer-gence inhibition and adulticidal activity of the leaf hexane,chloroform, ethyl acetate, acetone and methanol extracts ofAegle marmelos Correa ex Roxb, Andrographis lineataWallich ex Nees., Andrographis paniculata Wall. ex Nees.,Cocculus hirsutus Diels, E. prostrate and T. erecta were testedagainst Japanese encephalitis vector, Culex tritaeniorhynchus(Elango et al. 2012).
Shatavari is a perennial much branched climbing herbfound all over India, especially in tropical and subtropicalparts and in Himalayan region up to 1,400 m elevation. It isalso cultivated. The fleshy root of shatavari has been used asone of the most powerful nutritive and spermatogenic herb inAyurvedic system of medicine.
Shatavari (Asparagus racemosus) is an armed climbingunder shrub with woody terete stems and recurved and rarelystraight spines. Young stems very delicate, brittle and smooth;leaves reduced to minute chaffy scales and spines; cladodestriquetrous, curved in tufts or two to six. Flowers are white,fragrant in fascicles or racemes on the naked nodes of the mainshoots or in the axils of the thorns. Fruits are subglobose pulpyberries, purplish black when ripe. Seeds are three to six,globose or angled having brittle and hard testa. The tuberoussucculent roots are 30 cm to a metre or more in length,fascicled at the stem base and smooth tapering at both ends.Root contains saponin, water soluble constituents 52.1/2 %,moisture 1 %, glucose 7 % and ash from dried root 4 %. Theroots of Asparagus racemosus (Shatavari) are fleshy, whitish-brown in colour, lightly sweet in taste, emollient, cooling,nervine tonic and possesses rejuvenating, carminative andaphrodisiac properties. Different scientific studies haveproved its efficacy in a number of physical and mental ail-ments, respectively (Nadkarni 1976).
As far as our literature survey could ascertain, no informa-tion was available on ovicidal, larvicidal and adulticidal ac-tivities of the experimental plant species given. Therefore, themain aim of this study is to screen the mosquito ovicidal,larvicidal and adulticidal activities of crude extract fromAsparagus racemosus against Culex quinquefasciatus, Aedesaegypti and Anopheles stephensi (under laboratory condi-tions). This is the first report on the mosquito ovicidal, larvi-cidal and adulticidal activity of the solvent extracts of selectedplant.
Materials and methods
Collection of plants
The healthy root of Asparagus racemosus (Fig. 1) was col-lected fromKolli hills, Tamil Nadu, India. It was authenticatedby a plant taxonomist from the Department of Botany,Annamalai University, Annamalainagar. A voucher specimenwas deposited at the Department of Zoology, AnnamalaiUniversity.
Extraction
The healthy roots were washed with sterile distilled water,shade-dried and finely ground. The finely ground root powder
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(500 g/solvent) was extracted with five different solvents, viz,hexane, benzene, chloroform, ethyl acetate and methanolusing Soxhlet extraction apparatus, the extraction was contin-ued till visibly no further extraction is possible. 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 ovicidal,larvicidal and adulticidal bioassays.
Test organisms
The laboratory-bred pathogen-free strains of mosquitoes werereared in the vector control laboratory, Department of Zoology,Annamalai University. The larvae were fed on dog biscuits and
yeast powder in the 3:1 ratio. At the time of adult feeding, thesemosquitoes were 3–4 days old after emergences (maintained onraisins and water) and were starved for 12 h before feeding.Each time, 500 mosquitoes per cage were fed on blood using afeeding unit fitted with parafilm as membrane for 4 h. Aedesaegypti feeding was done from 12 noon to 4.00 p.m. andAnopheles stephensi and Culex quinquefasciatuswere fed dur-ing 6.00 p.m. to 10.00 p.m. Amembrane feeder with the bottomend fitted with parafilm was placed with 2.0 ml of the bloodsample (obtained from a slaughter house by collecting in aheparinized vial and stored at 4 °C) and kept over a netted cageof mosquitoes. The blood was stirred continuously using anautomated stirring device, and a constant temperature of 37 °Cwere maintained using a water jacket circulating system. Afterfeeding, the fully engorged females were separated and main-tained on raisins. Mosquitoes were held at 28±2 °C, 70–85 %relative humidity, with a photo period of 12-h light and 12-hdark.
Ovicidal activity
For ovicidal activity, slightly modified method of Su andMulla (1998) was performed. Anopheles stephensi, Aedesaegypti and Culex quinquefasciatus eggs were collectedfrom vector control laboratory, Department of Zoology,Annamalai University. The root extracts were diluted inthe ethanol to achieve various concentrations ranging from75 to 450 ppm. Eggs of these mosquito species (100) wereexposed to each concentration of leaf extracts. After 24 htreatment, the eggs from each concentration were individu-ally transferred to distilled water cups for hatching assess-ment after counting the eggs under the microscope. Eachexperiment was replicated six times along with appropriatecontrol. The hatch rates were assessed 48 h post-treatmentby following formula:
% hatchability ¼ Number of hatched larvae
Total number of eggs� 100
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 a 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 (40–300 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 (lethal
Fig. 1 Asparagus racemosus plant
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concentration 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).
Adulticidal bioassay
Adulticidal bioassay was performed by WHO method(1981). Appropriate concentrations of crude extracts werediluted with ethanol to achieved different concentrationsand applied on Whatman no. 1 filter papers (size 12×15 cm). Control papers were treated with ethanol undersimilar conditions. Adulticidal activity of the crude extractswas evaluated at various concentrations (from 50 to400 ppm). Twenty female mosquitoes were collected andgently transferred into a plastic holding tube. The mosqui-toes were allowed to acclimatize in the holding tube for1 h and then exposed to test paper for 1 h. At the end ofexposure period, the mosquitoes were transferred back tothe holding tube and kept 24 h for recovery period. A padof cotton soaked with 10 % glucose solution was placedon the mesh screen. Each experiment was replicated fivetimes along with appropriate control. Mortality of mosqui-toes was determined at the end of 24 h recovery period.LD50, LD90 with their 95 % confidence limits were deter-mined using Log probit analysis test (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, slope,regression equation and chi square test were calculated usingthe SPSS14.0 (Statistical Package of Social Sciences Inc.,USA) software.
Results
The mean percent egg hatchabilities of Culex quinquefasciatus,Aedes aegypti and Anopheles stephensi were tested with fivedifferent solvents at different concentrations of Asparagusracemosus root extracts, and the results are listed in Table 1.The percent hatchability was inversely proportional to the con-centration of extract and directly proportional to the eggs.Among the extracts tested for ovicidal activity against Culexquinquefasciatus, Aedes aegypti and Anopheles stephensi, themethanol extract of Asparagus racemosus exerted 100 % mor-tality (zero hatchability) at 375, 300 and 225 ppm, respectively.Control eggs showed above 99.0–100%hatchability. The resultsof the larvicidal activity of crude hexane, benzene, chloroform,ethyl acetate and methanol solvent leaf extracts of Asparagusracemosus against the larvae of three important vector
Table 1 Ovicidal activity of different solvent root extracts of Asparagus racemosus against Anopheles stephensi, Culex quinquefasciatus and Aedesaegypti
mosquitoes viz. Culex quinquefasciatus, Aedes aegypti andAnopheles stephensi are presented in Tables 2, 3, 4, 5 andFig. 2a–c. Among the extracts tested, the highest larvicidalactivity was observed in methanol extract against Anophelesstephensi followed by Aedes aegypti and Culex quinquefasciatuswith the LC50 and LC90 values of 90.97, 97.71 and 115.13 ppmand 168.82, 179.92, and 210.96 ppm, respectively. The 95 %confidence limits LC50 (LCL-UCL) and LC90 (LCL-UCL), re-gression equation, slope and chi square values were also calcu-lated. The results of the adulticidal activity of hexane, benzene,chloroform, ethyl acetate and methanol solvent leaf extracts ofAsparagus racemosus against the adult of three important vectormosquitoes, viz., Culex quinquefasciatus, Aedes aegypti and
Anopheles stephensi, are presented in Tables 6, 7, 8, 9 andFig. 3a–c. The plant crude extracts showed dose-dependentmortality. At higher concentrations, the adult showed restlessmovement for some timeswith abnormal wagging and then died.Among the extracts tested, the highest adulticidal activity wasobserved in methanol extract against Culex quinquefasciatus,Aedes aegypti and Anopheles stephensi. The LD50 and LD90
values of Asparagus racemosus root extracts against adulticidalactivity of (hexane, benzene, chloroform, ethyl acetate and meth-anol) Culex quinquefasciatus, Aedes aegypti and Anophelesstephensiwere the following: Anopheles stephensi, LD50 values157.16, 143.49, 154.68, 133.43 and 120.44 ppm, and LD90
values were 282.57, 254.28, 272.87, 241.19 and 214.65 ppm;
Table 2 Percentage mortality of mosquito larvae of Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi exposed to different concentrationsof different solvent leaf crude extracts of Asparagus racemosus
Solvents Culex quinquefasciatus Aedes aegypti Anopheles stephensi
Concentration (ppm) % of mortality±SDa Concentration (ppm) % of mortality±SDa Concentration (ppm) % of mortality±SDa
Hexane Control 0±0.0 Control 0±0.0 Control 0±0.0
60 24±0.8 60 28±0.8 50 24±0.8
120 42±1.3 120 46±1.3 100 41±1.5
180 61±0.9 180 65±0.9 150 59±0.9
240 77±0.9 240 82±1.3 200 78±1.3
300 94±1.3 300 99±0.5 250 94±1.3
Benzene Control 0±0.0 Control 0±0.0 Control 0±0.0
60 32±0.8 50 28±0.8 50 31±0.9
120 49±0.9 100 47±0.9 100 49±0.9
180 67±0.9 150 62±1.3 150 65±0.9
240 84±0.8 200 81±1.3 200 83±0.9
300 99±0.5 250 97±0.9 250 99±0.5
Chloroform Control 0±0.0 Control 0±0.0 Control 0±0.0
60 29±1.5 50 24±0.8 50 27±0.9
120 47±0.9 100 42±1.3 100 45±0.9
180 65±0.9 150 59±0.9 150 62±1.3
240 80±0.8 200 78±1.3 200 80±1.4
300 97±0.9 250 94±1.3 250 97±0.9
Ethyl acetate Control 0±0.0 Control 0±0.0 Control 0±0.0
50 26±1.3 50 30±1.3 40 26±1.3
100 45±0.9 100 49±0.9 80 45±0.9
150 61±0.9 150 68±0.9 120 62±1.3
200 78±1.3 200 83±0.9 160 79±0.9
250 96±0.8 250 99±0.5 200 97±0.9
Methanol Control 0±0.00 Control 0±0.0 Control 0±0.0
50 28±0.8 40 25±0.9 40 29±0.9
100 47±0.9 80 46±1.3 80 48±0.8
150 65±0.9 120 62±1.3 120 66±1.3
200 81±0.9 160 79±0.9 160 82±1.3
250 99±0.5 200 95±1.3 200 98±0.6
SD standard deviationa Values are mean±SD of four replicates
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Aedes aegypti, LD50 values were 177.69, 162.52, 172.92, 143.14and 135.60 ppm, and LD90 values were 322.30, 296.84, 315.30,257.76 and 248.35 ppm; and Culex quinquefasciatus LD50
values were 208.46, 186.22, 196.36, 166.57 and 157.71 ppm,and LD90 values were 368.22, 333.71, 350.12, 303.44 and290.95 ppm, respectively. No mortality was recorded in thecontrol. The 95 % confidence limits LD50 (LCL-UCL) andLD90 (LCL-UCL), slope, regression equation and chi square testwere also calculated.
Discussion
Plants are rich sources of bioactive compounds that can be usedto develop environmentally safe vector and pest managingagents. Phyto-extracts are emerging as potential mosquito con-trol agents, with low-cost, easy-to-administer and risk-freeproperties. Simple crude extracts from plants have been usedas insecticides in many countries for centuries (Crobsy et al.1971). Berenbaum (1985) stated that the crude plant extractsoften consist of complex mixtures of active compounds.Advances of using complete mixture may act synergistically,they may show greater overall bioactivity compared to theindividual constituents (Chen et al. 1995), respectively. Thescreening of local medicinal plants for mosquito larvicidaland ovicidal activity may eventually lead to their use in naturalproduct-based mosquito abatement practices (Bowers et al.1995). Our result showed that the crude hexane, benzene,chloroform, ethyl acetate and methanol solvent extracts of root
of Asparagus racemosus have significant ovicidal, larvicidaland adulticidal properties against three important vector mos-quitoes viz., Culex quinquefasciatus, Aedes aegypti andAnopheles stephensi. This result is also comparable to earlierreports of Mullai et al. (2008) who reported that the benzeneextracts of Citrullus vulgaris exerted 100 % mortality (zerohatchability) at 250 ppm, a very low hatchability (11.8 %) at200 ppm, complete ovicidal activity at 300 ppm and the fractionI at 80 ppm exerted a very low hatchability rate of 3.2 %followed by fraction II (6.9 %), and fraction III and fractionIV which afforded 4.9 and 5.3 % hatchability recorded againstAnopheles stephensi and Aedes aegypti, respectively. The ovi-cidal effect of Solenostemma argelwas low; however, concen-trations of 0.05 and 0.1 % exhibited significant effects(p<0.05), producing 65 and 75 %, and 62.9 and 62.9 %,respectively, on the first and second day after treatment, respec-tively; the 0.1 % concentration reduced egg hatch by 33.7 %,compared with the control, and 100 % mortality values wereevident in concentrations as low as 0.025 % at 2 days post-hatching against Culex pipiens (Al-Doghairi et al. 2004). Theseed extract of Atriplex canescens showed complete ovicidal at1,000 ppm concentration in eggs of Culex quinquefasciatus(Ouda et al. 1998). Rahuman and Venkatesan (2008) reportedlarvicidal activity ofmethanolic leaf extracts ofCoccinia indica,Momordica charantia, Trichosanthes anguina and Cucumissativus against Aedes aegypti, i.e., LC50=309.46, 199.14,554.20 and 492.73 ppm, respectively. They have also reportedlarvicidal activity of the same plant extracts against Culexquinquefasciatus, i.e., LC50=377.69, 207.61, 842.34 and
Table 3 LC50, LC90, slope, regression equation and chi square analysis of larvicidal activity of Asparagus racemosus against Culex quinquefasciatus
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at p<0.05
Parasitol Res
623.80 ppm, respectively. Marimuthu et al. (2011) reportedbioactivity of synthesized AgNPs against the larvae ofAnopheles subpictus, Culex quinquefasciatus andRhipicephalus microplus (LC50=13.90, 11.73 and 8.98 ppm),respectively.
The biological remedies for mosquito control includes theuse of plant extract and plant essential oils as an effectiverepellent, ovocides, larvicides and adulticides; however, theefficacy of plant extract varies from species to species and plantparts (Shaalan et al. 2005). Mullai and Jebanesan (2006) re-ported that the complete ovicidal activity (100%mortality) wasattained at 300 ppm for methanol, benzene, petroleum ether andethyl acetate extracts of Citrullus pubescens against Culexquinquefasciatus. The leaf extract of Solanum trilobatum re-duced egg laying by gravid females of Anopheles stephensifrom 18 to 99% comparedwith ethanol-treated controls at 0.01,0.025, 0.05, 0.075 and 0.1 % (Rajkumar and Jebanesan 2005).Bagavan et al. (2008) have reported peel chloroform extract ofCitrus sinensis, leaf ethyl acetate extracts of Ocimum canumand Ocimum sanctum, and leaf chloroform extract ofRhinacanthus nasutusagainst the larvae of Anopheles subpictus(LC50=58.25, 88.15, 21.67 and 40.46 ppm; LC90=298.31,528.70, 98.34 and 267.20 ppm) and peel methanol extract ofCitrus sinensis, leaf methanol extract of O. canum and ethylacetate extracts of O. sanctum and R. nasutus against the larvaeof Culex tritaeniorhynchus (LC50=38.15, 72.40, 109.12 and39.32 ppm; LC90=184.67, 268.93, 646.62 and 176.39 ppm),respectively. Kamaraj et al. (2008) reported that the highestlarval mortality was found in leaf petroleum ether, flowermethanol extracts ofCryptocoryne auriculata, flower methanolextracts of L. aspera and R. nasutus, leaf and seed methanolextracts of Solanum torvum, and leaf hexane extract of Vitexnegundo against the larvae of Anopheles subpictus (LC50=44.21, 44.69, 53.16, 41.07, 35.32, 28.90 and 44.40 ppm;LC90=187.31, 188.29, 233.18, 142.66, 151.60, 121.05 and192.11 ppm, respectively) and against the larvae of Culextritaeniorhynchus (LC50=69.83, 51.29, 81.24, 71.79, 44.42,84.47 and 65.35 ppm; LC90=335.26, 245.63, 300.45, 361.83,185.09, 351.41 and 302.42 ppm, respectively). Ansari et al.(2005) observed the larvicidal activity of Pinus longifolia oilagainst three vector mosquitoes namely Aedes aegypti (LC50,
Rajkumar and Jebanesan (2004) studied ovicidal activity ofMoschosma polystachyum leaf extract against Culexquinquefasciatus and observed 100 % egg mortality at100 ml/l. Larvicidal activity of crude extract of Sida acutaagainst Culex quinquefasciatus, Aedes aegypti and Anophelesstephensi with LC50 values ranging between 38 and 48 mg/l(Govindarajan 2010) The ovicidal effect of Solenostemmaargel was low; however, concentrations of 0.05 and 0.1 %exhibited significant effects (p<0.05), producing 65 and 75 %and 62.9 and 62.9 %, respectively, on the first and second dayafter treatment, respectively. The 0.1 % concentration reducedegg hatch by 33.7 %, compared with the control, and 100 %mortality values were evident in concentrations as low as0.025 % at 2 days post-hatching against Culex pipiens (Al-Doghairi et al. 2004). The seed extract of Atriplex canescensshowed complete ovicidal at 1,000 ppm concentration in eggsof Culex quinquefasciatus (Ouda et al. 1998). The bioactivecompound Azadirachtin isolated from Azadirachta indicashowed complete ovicidal activity in eggs of Culex tarsalisand Culex quinquefasciatus exposed to 10 ppm concentration(Ouda et al. 1998).
Earlier authors reported that the methanol leaf extracts ofV. negundo, Vitex trifolia, Vitex peduncularisand Vitex altissimawere used for larvicidal assay with LC50 value of 212.57,41.41, 76.28 and 128.04 ppm, respectively, against the earlyfourth-instar larvae of Culex quinquefasciatus (Kannathasanet al. 2007). The same extracts ofEuphorbia tirucalli latex andstem bark were evaluated for larvicidal activity againstlaboratory-reared larvae of Culex quinquefasciatus withLC50 values of 177.14 and 513.387 mg/l, respectively(Yadav et al. 2002). Sharma et al. (2005) reported that theacetone extract of Nerium indicum and Thuja orientelis hasbeen studied with LC50 values of 200.87, 127.53, 209.00 and155.97 ppm against third-instar larvae of Anopheles stephensiand Culex quinquefasciatus. Larvicidal activity of the acetoneextracts of Murraya koenigii, Coriandrum sativum, Ferulaasafoetida and Trigonella foenum graceum was tested outusing different concentrations of each plant (range, 25–900 ppm) against Aedes aegypti larvae (Harve and Kamath
Table 5 LC50, LC90, slope, regression equation and chi square analysis of larvicidal activity of Asparagus racemosus against Anopheles stephensi
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at p<0.05
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2004); Rahuman and Venkatesan (2008) reported the petro-leum ether extract of Citrullus colocynthis; methanol extractsof Cannabis indica, Cannabis sativus and Momordicacharantia; and acetone extract of Trichosanthes anguinaagainst the larvae of Aedes aegypti (LC50=74.57, 309.46,492.73, 199.14 and 554.20 ppm) and against Culexquinquefasciatus (LC50=88.24, 377.69, 623.80, 207.61 and842.34 ppm), respectively. Larvicidal efficacies of methanolextracts of Momordica charantia, Trichosanthes anguina,
Luffa acutangula, Benincasa cerifera and Citrullus vulgaristested with LC50 values were 465.85, 567.81, 839.81,1,189.30 and 1,636.04 ppm, respectively, against the late thirdlarval age group of Culex quinquefasciatus (Prabakar andJebanesan 2004). Malarvanan et al. (2009) reported thatCipadessa baccifera, Melia dubia, Clausena dentata, andDodonaea angustifolia of petroleum ether, hexane, chloro-form, acetone, and water extracts exhibited ovicidal activityagainst Helicoverpa armigera and maximum activity was
LC50
LC90
LC50
LC90
LC50
LC90
a
b
c
Fig. 2 a–cGraph showing theLC50 and LC90 values of threeimportant mosquito larvae
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Table 6 Percentagemortality ofmosquito adult ofCulex quinquefasciatus,Aedes aegyptiand Anopheles stephensiexposed to different concentrations ofdifferent solvent leaf crude extracts of Asparagus racemosus
Solvents Culex quinquefasciatus Aedes aegypti Anopheles stephensi
Concentration (ppm) % of mortality±SDa Concentration (ppm) % of mortality±SDa Concentration (ppm) % of mortality±SDa
Hexane Control 0±0.0 Control 0±0.0 Control 0±0.0
80 21±0.8 80 29±0.8 70 26±0.8
160 38±0.5 160 46±1.3 140 48±1.1
240 61±0.4 240 69±0.8 210 69±0.8
320 76±0.4 320 84±0.8 280 83±0.8
400 95±0.7 400 100±0.0 350 100±0.0
Benzene Control 0±0.0 Control 0±0.0 Control 0±0.0
80 28±0.5 70 28±0.5 60 21±0.8
160 42±1.3 140 46±0.8 120 47±1.3
240 65±0.7 210 64±0.8 180 65±0.7
320 82±1.1 280 81±0.8 240 81±0.8
400 100±0.0 350 99±0.4 300 98±0.5
Chloroform Control 0±0.0 Control 0±0.0 Control 0±0.0
80 25±1.2 70 25±0.7 60 19±0.8
160 40±1.2 140 44±0.8 120 42±0.8
240 63±0.5 210 61±0.8 180 61±0.8
320 79±0.8 280 78±0.5 240 77±0.5
400 98±0.5 350 96±0.8 300 95±0.7
Ethyl acetate Control 0±0.0 Control 0±0.0 Control 0±0.0
70 25±1.0 60 25±0.7 60 27±0.5
140 46±0.8 120 45±1.0 120 49±0.8
210 65±0.7 180 63±0.5 180 68±0.8
280 80±0.7 240 81±0.8 240 84±0.8
350 97±0.5 300 98±0.5 300 100±0.0
Methanol Control 0±0.0 Control 0±0.0 Control 0±0.0
70 29±0.8 60 29±1.3 50 22±0.5
140 48±1.1 120 48±1.5 100 47±1.1
210 67±0.5 180 65±1.0 150 64±0.8
280 82±1.1 240 82±0.5 200 78±0.8
350 99±0.4 300 100±0.0 250 99±0.4
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 Asparagus racemosusagainst Culex quinquefasciatus
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at p<0.05
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observed in hexane extract of Clausena dentata. Mullai andJebanesan (2006) reported that the complete ovicidal activity(100 % mortality) was attained at 300 ppm for ethanol, ben-zene, petroleum ether and ethyl acetate extracts of Citrulluspubescens against Culex quinquefasciatus. The 100 % ovicid-al activity of the Cymbopogon citratus oil against the adultmosquitoes Culex quinquefasciatus and Aedes aegypti hasbeen revealed by Pushpanathan et al. (2006b) at 300 ppm.
Komalamisra et al. (2005) have reported that the petroleumether and methanol (MeOH) extracts of R. nasutus and Derriselliptica exhibited larvicidal effects against Aedes aegypti,Culex quinquefasciatus, Anopheles dirus and Mansoniauniformiswith LC50 values between 3.9 and 11.5 mg/l, whilethe MeOH extract gave LC50 values of between 8.1 and14.7 mg/l. D. elliptica petroleum ether extract showed LC50
values of between 11.2 and 18.84 mg/l, and the MeOH extractexhibited LC50 values between 13.2 and 45.2 mg/l. The n-hexane, ethyl acetate and methanol extracts of Cassianigricans showed 100 % larval mortality againstOchlerotatus triseriatus (Georges et al. 2008). Jang et al.(2002) have reported that the methanol extracts of Cassiaobtusifolia, Cassia tora and Vicia tetrasperma exhibited morethan 90 % larval mortality at 200 ppm on Aedes aegypti andCulex pipiens. The larvicidal activity of petroleum ether,ethanolic, aqueous extracts of dried leaves and fixed oil fromthe seeds of Caesalpinia bonduc (Family: Caesalpiniaceae)
showed 100 % mortality in 1 % concentration of petroleumether and ethanolic extract of leaf, whereas it was 55 % in2.5 % concentration of aqueous extract and 92.6 % in 2.5 %concentration of fixed oil against the fourth-instar larvae ofCulex quinquefasciatus (Saravanan et al. 2007); the petroleumether extract of Solanum xanthocarpum was observed to bethe most toxic with LC50 of 1.41 and 0.93 ppm and LC90 of16.94 and 8.48 ppm at 24 and 48 h after application, respec-tively, against Anopheles stephensi (Mohan et al. 2007).
The adulticidal and repellent activities of crude hexane,ethyl acetate, benzene, chloroform and methanol extracts ofleaf of E. alba and Andrographis paniculatawere assayed fortheir toxicity against two important vector mosquitoes, viz.,Culex quinquefasciatus and Aedes aegypti. The highest adultmortality was found in methanol extract of Andrographispaniculata against the adults of Culex quinquefasciatus andAedes aegypti with the LC50 and LC90 values of 149.81 and172.37 ppm and 288.12 and 321.01 ppm, respectively(Govindarajan and Sivakumar 2012a). Jeyabalan et al.(2003) also have reported the adulticidal effect ofPelargonium citrosa on Anopheles stephensi, with LC50 andLC90 value of 1.56 and 5.22 %, respectively. However, it isworth to note that their LC50 and LC90 values were muchhigher than the extracts, which were tested in this study.Corbel et al. (2004) demonstrated the excellent intrinsic tox-icity, measured by topical application against laboratory-
Table 8 LD50, LD90, slope, regression equation and chi square analysis of adulticidal activity of different solvent leaf extracts of Asparagus racemosusagainst 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 Asparagus racemosusagainst Anopheles stephensi
LCL lower confidence limits, UCL upper confidence limits, χ2 chi square, df degrees of freedoma Significant at p<0.05
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reared Aedes aegypti adults, of some pyrethroids and organo-phosphates, including bifenthrin, permethrin and temephos,with an LD50 value of 0.077, 0.24 and 195 ng/mg female,respectively. The larvicidal and adulticidal activities ofethanolic and water mixture (50:50) of plant extractsEucalyptus globulus, Cymbopogan citratus, Artemisia annua,Justicia gendarussa, Myristica fragrans, Annona squamosa
and Centella asiaticawere tested against Anopheles stephensi,and the most effective between 80 and 100 % was observed inall extracts (Senthilkumar et al. 2009).
Halim (2008) have reported the insecticidal activity ofZingiber officinale against the larval maturation, and adultemergency ofAnopheles pharoensis third stage was evaluated:the concentrations of 100, 70, 50, 25, 5, 2, 1, 0.9, 0.7, 0.5 and
LD50
LD90
LD50
LD90
LD50
LD90
a
b
c
Fig. 3 a–cGraph showing theLD50 and LD90 values of threeimportant adult mosquitoes
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0.3 % showed 100 % larval mortality rate and at 0.2 and 0.1 %caused mortality of 66.7 %, respectively. Dua et al. (2010)observed the adulticidal activity of the essential oil of Lantanacamara evaluated against different mosquitoes species on0.208 mg/cm2 impregnated papers, the KDT50 and KDT90values of the essential oil were 20, 18, 15, 12 and 14 min and35, 28, 25, 18 and 23 min against Aedes aegypti, Culexquinquefasciatus, Anopheles culicifacies, Anopheles fluvialitisand Anopheles stephensi with their percent mortality of 93.3,95.2, 100, 100 and 100 %, respectively. The ethanol extract ofApium graveolence exhibited adulticidal activity against Aedesaegypti with LD50 and LD90 values of 6.6 and 66.4 mg/cm2
(Choochote et al. 2004). The root extract of Valeriana jatamansiwhich exhibited adulticidal activity of 90 % lethal concentrationagainst adult Anopheles stephensi, Anopheles culicifacies, Aedesaegypti, Aedes albopictus and Culex quinquefasciatuswas 0.14,0.16, 0.09, 0.08 and 0.17, and 0.24, 0.34, 0.25, 0.21 and0.28 mg/cm2, respectively (Dua et al. 2008). The unsaponifiableportion and volatile oil of Thymus capitatus showed the highestadulticidal potency (LC50=0.0070 and 0.0076 mg/cm2, respec-tively, against Culex pipiens) (Mansour et al. 2000). Priyankaand Prakash (2003) have also tested theChrysosporium tropicummetabolite against mosquito larvae of Anopheles stephensi andthe third-instars larvae of Culex quinquefasciatus, respectively.They reported that first-instars larvae were most susceptible withLC50 value of 38.9 μl/ml compared with fourth instar, which is122.6 μl/ml. The LC50 value of alkali-solubilized parasporalinclusion proteins of a Diptera-specific strain, belonging toBacillus thuringiensis serovar canadensis, was 2.4 μg/ml forlarvae of the mosquito, Aedes aegypti. A significant loss inlarvicidal activity occurred when solubilized inclusion proteinswere treated with Aedes aegypti larval gut extract, silkworm(Bombyx mori) larval gut juice and the proteinase K.Approximately 90 % of the larvicidal activity was destroyedupon treatment with proteases in 30 min. The parasporal inclu-sion was composed of major proteins of 65, 53 and 28 kDa andsome other minor proteins. The bioactive compoundAzadirachtin isolated from Azadirachta indica showed completeovicidal activity in eggs of Culex tarsalis and Culexquinquefasciatus exposed to 10 ppm concentration (Su andMulla 1998).
In conclusion, our study reveals that the crude extract ofAsparagus racemosus has remarkable ovicidal, larvicidal andadulticidal properties. The flora of India has rich aromaticplant diversity with potential for development of natural in-secticides for control of mosquito and other pests. In brief, ourfindings suggested that the crude extract from Asparagusracemosus root may be explored as a potential environmentalbenign mosquitocide. Further investigations for the mode ofthe constituents’ actions, effects on nontarget organisms andfield evaluation are necessary. These results obtained areuseful in search of more selective, biodegradable and naturallyproduced mosquitocidal compounds.
Acknowledgments The authors are grateful to Indian Council of Med-ical Research (ICMR Ref. Letter No. 5/8-7 (246)/2012-ECD-II), NewDelhi, India, for providing financial assistance and the authors would liketo thank Dr. 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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