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Mosquito larvicidal and pupicidal activity of Euphorbia hirta Linn. (Family:

Euphorbiaceae) and Bacillus sphaericus against Anopheles stephensi Liston.
(Diptera: Culicidae)

Article  in  Asian Pacific Journal of Tropical Medicine · February 2013

DOI: 10.1016/S1995-7645(13)60003-6 · Source: PubMed

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 Asian Pacific Journal of Tropical Medicine (2012)412-420 1

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Medicine

journal homepage:www.elsevier.com/locate/apjtm

Document heading doi:

Mosquito larvicidal and pupicidal activity of Euphorbia hirta Linn. (Family:

Euphorbiaceae) and Bacillus sphaericus against Anopheles stephensi Liston.
(Diptera: Culicidae)
C. Panneerselvam1, K. Murugan2, K. Kovendan2*, P. Mahesh Kumar2, J. Subramaniam2
1DRDO-BU Center for Life Sciences, Bharathiar University, Coimbatore - 641 046, Tamil Nadu, India
1

Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore - 641 046, Tamil Nadu, India
2

ARTICLE INFO ABSTRACT

Article history: Objective: To explore the larvicidal and pupicidal activity of Euphorbia hirta (E. hirta) leaf
Received 15 November 2012
extract and Bacillus sphaericus (B. sphaericus) against the malarial vector, Anopheles stephensi (An.
Received in revised form 27 December
stephensi). Methods: The larvicidal and pupicidal activity was assayed against An. stephensi at
2012
Accepted 28 January 2013 various concentrations ranging from (75-375 ppm) under the laboratory as well as field conditions.
Available online 28 February 2013 The LC50 and LC90 value of the E. hirta leaf extract was determined by probit analysis. Results:
The plant extract showed larvicidal effects after 24 h of exposure; however, the highest larval
mortality was found in the methanol extract of E. hirta against the first to fourth instars larvae
Keywords: and pupae of values LC50= 137.40, 172.65, 217.81, 269.37 and 332.39 ppm; B. sphaericus against the
Euphorbia hirta first to fourth instars larvae and pupae of values LC50= 44.29, 55.83, 68.51, 82.19 and 95.55 ppm,
Anopheles stephensi respectively. Moreover, combined treatment of values of LC50= 79.13, 80.42, 86.01, 93.00 and 98.12
Bacillus sphaericus ppm, respectively. No mortality was observed in the control. Conclusions: These results suggest
Larvicidal and pupicidal activity methanol leaf extracts of E. hirta and B. sphaericus have potential to be used as an ideal eco-
Field trial friendly approach for the control of the malarial vector, An. stephensi as target species of vector
Malarial vector control programs. This study provides the first report on the combined mosquito larvicidal and
pupicidal activity of this plant crude extract and bacterial toxin against An. stephensi mosquitoes.

drugs, malaria control in the developing countries is based

1. Introduction largely on vector eradication by the application of mosquito
larvicides as an ideal method for controlling mosquito
Malaria is one of the serious scourges inflicted upon infestation with malaria parasites. Among 53 anopheline
humanity. It causes human mortality and morbidity along species present in India, nine are vectors malaria. Anopheles
with great financial loss. Almost all tropical regions of the stephensi (An. stephensi) is responsible for transmission of
world are experiencing the resurgence and reoccurrence malaria in urban regions of India[1].
of one of the world’s most deadly diseases, ie. malaria, and An obvious method for the control of mosquito-borne
India is no exception. In the Indian scenario, almost the diseases is the use of insecticides, and many synthetic
entire country is endemic to the disease due to favorable agents have been developed and employed in the field
ecological conditions. The incidence of malaria in the with considerable success. However, one major drawback
country is largely erratic regionally because of various with the use of chemical insecticides is that they are non-
biological and climatic factors. Further, malaria exerts selective and could be harmful to other organisms in the
an enormous toll in terms of medical cost and in days of environment. It has also provoked undesirable effects,
labor cost in the country. Besides the use of antimalarial including toxicity to nontarget organisms, and fostered
environmental and human health concerns[2]. The toxicity
*Corresponding author: Dr. K. Kovendan, Division of Entomology, Department of problem, together with the growing incidence of insect
Zoology School of Life Sciences, Bharathiar University, Coimbatore-641 046, India. resistance, has called attention to the need for novel
Tel: +91-9962447932
E-mail: [email protected]
insecticides[3] and for more detailed studies of naturally
2 K. Kovendan et al./Asian Pacific Journal of Tropical Medicine (2013)412-420

occurring insecticides[4]. These problems have highlighted sold under the trade name Vectolex. When community
the need for the development of new strategies for selective mosquito control is needed to reduce mosquito-borne
mosquito larval control. Extracts or essential oils from plants disease, the Department of Health favors the use of
may be alternative sources of mosquito larval control agents, larvicide applications targeted to the breeding source of
as they constitute a rich source of bioactive compounds that mosquitoes[24]. It produces a round spore in the terminal
are biodegradable into nontoxic products and potentially portion of its cell. Two types of proteins, crystal toxins
suitable for use in control of mosquito larvae. In fact, many and Mtx toxins, produce the larvicidal effect by acting on
researchers have reported on the effectiveness of plant specific receptors in the midgut of culicid larvae, causing
extracts or essential oils against mosquito larvae[5-7]. a lethal cytopathological effect. Despite its high toxicity, B.
Euphorbia hirta (E. hirta) belongs to the family sphaericus is very specific, affecting only a small number
Euphorbiaceae. It is a small annual herb common to of susceptible species. Another important factor is the
tropical countries. It is usually erect, slender-stemmed; bioinsecticide’s capacity to recycle itself in dead Culicidae
spreading up to 80 cm tall, though sometimes it can be larvae, resulting in greater persistence and greater larvicidal
seen lying down. The plant is an annual broad-leaved herb effect by the product. Based on such characteristics, B.
that has a hairy stem with many branches from the base to sphaericus bioinsecticides are indicated for combating
top. The leaves are opposite, elliptical, oblong or oblong- lymphatic filariasis, West Nile fever, and malaria among
lanceolate, with a faintly toothed margin and darker on the other diseases.
upper surface. The flower are small, numerous and crowded Many biological control agents have been evaluated
together in dense cymes (dense clusters in upper axils) about against larval stages of mosquitoes, of which the most
1 cm in diameter. The stem and leaves produce a white or successful ones comprise bacteria such as Bacillus
milky juice when cut. It is frequently seen occupying open thuringiensis (B. thuringiensis) and B. sphaericus[25]. The
waste spaces, banks of watercourses, grasslands, road sides, use of B. sphaericus as a potential biolarvicides in India is
and pathways[8,9]. limited due to the development of resistance by the target
The genus Euphorbia (Euphorbiaceae) is chemically mosquito species[26]. Well-known bacterial agents which
defined by the occurrence of a large number of have been used successfully for mosquito control are B.
polyfunctional diterpenoids with the tigliane (phorbol), thuringiensis and B. sphaericus. Two bacterial agents such as
ingenane, and daphnane skeletons [10] ; lectins and the B. thuringiensis and B. sphaericus are being widely used
lysozymes with recognized biological properties [11,12]. for control of mosquito breeding in a variety of habitats[27-
Most of these are skin irritants and toxic; in addition, 31]. The mosquitocidal activity of the highly active strain of
many of them are skin tumor promoters. Nonirritant B. sphaericus resulted in their development as a commercial
polyfunctional macrocyclic diterpenoids with the lathyrane larvicides. This is now used in many countries in various
and jatrophane skeletons have also been isolated from the parts of the world to control vector and nuisance mosquito
Euphorbia species. The plant has been reported to contain species[32].
quercitrin[13] and polyphenols[14]. The extracts were reported The present investigation was to explore the mosquito
as anthelmintic[15], repellent, antifeedant and controlling control agent under laboratory as well as field conditions.
Plutella xylostella [16] and Rotylenchulus reniformis [17], The plant extracts and B. sphaericus are reported to have
antimicrobial[18] antibacterial, and against worms[19]. mosquitocidal properties against malarial vector, An.
Many studies on plant extracts against mosquito larvae stephensi as target species.
have been conducted around the world. Larvicidal activity
of Gliricidia sepium crude ethanol extracts of dried leaves,
fresh leaves, dried petioles and stem bark were tested for 2. Materials and methods
their activities against third instar larvae of An. stephensi,
Anopheles aegypti and Culex quinquefasciatus[20]. Studies 2.1. Collection of eggs and maintenance of larvae
were focused on the effect of some indigenous plants on the
larvicide and ovipositional properties on An. stephensi[21]. The eggs of An. stephensi were collected from National
The petroleum ether root extract of Solanum xanthocarpum Centre for Disease Control field station of Mettupalayam,
extract was observed to be toxic against the larvae of An. Tamil Nadu, India, using an “O”-type brush. These eggs
stephensi[22]; the leaf extract of Solanum trilobatum was were brought to the laboratory and transferred to 18 cm暳
tested under laboratory conditions for oviposition-deterrent 13 cm暳4 cm enamel trays containing 500 mL of water for
and skin-repellent activities against An. stephensi[23]. hatching. The mosquito larvae were pedigree dog biscuits
Bacillus sphaericus (B. sphaericus) is a naturally occurring and yeast at 3:1 ratio. The feeding was continued until the
soil bacterium that can effectively kill mosquito larvae larvae transformed into the pupal stage.
present in water. B. sphaericus has the unique property of
being able to control mosquito larvae in water that is rich in 2.2. Maintenance of pupae and adults
organic matter. B. sphaericus is effective against Culex spp.
but is less effective against some other mosquito species. The pupae were collected from the culture trays and
Commercially available formulations of B. sphaericus are transferred to plastic containers (12 cm伊12 cm) containing
 K. Kovendan et al./Asian Pacific Journal of Tropical Medicine (2013)412-420
3

500 mL of water with the help of a dipper. The plastic jars and B. sphaericus were added. Larval food was given for the
were kept in a 90 cm暳 90 cm暳 90 cm mosquito cage for test larvae. At each tested concentration two to five trials
adult emergence. Mosquito larvae were maintained at (27 were made and each trial consisted of five replicates. The
依2) 曟, 75%-85% relative humidity, under a photoperiod of control was setup by mixing 1 mL of acetone with 249 mL
14:10 (light/dark). A 10% sugar solution was provided for a of dechlorinated water. The larvae and pupae were exposed
period of 3 days before blood feeding. to dechlorinated water without acetone served as control.
The control mortalities were corrected by using Abbott's
2.3. Blood feeding of adult An. stephensi formula[34]:
Observed mortality in treatment-
The adult female mosquitoes were allowed to feed on the Observed mortality in control 伊100
Corrected mortality=
blood of a rabbit (a rabbit per day, exposed on the dorsal 100-Control mortality
side) for 2 days, to ensure adequate blood feeding for 5 days.
After blood feeding, enamel trays with water from the culture
trays were placed in the cage as oviposition substrates. Number of dead larvae/pupae
Percented mortality= 伊100
Number of larvae/pupae introduced
2.4. Collection of plant and preparation of extract

The E. hirta plants were collected from in and around

Bharathiar University Campus, Coimbatore. The plants were The LC50 and LC90 were calculated from toxicity data by
identified at Botanical Survey of India, Coimbatore, India. using probit analysis[35].
E. hirta leaves were washed with tap water and shade dried
at room temperature. The dried plant materials (leaves) were 2.7. Field trail
powdered by an electrical blender. From the powder 500
g of the plant material were extracted with 1.5 L of organic For the field trial, the quantity of plant extract residues and
solvents of methanol for using a Soxhlet apparatus boiling B. sphaericus (Bs) required (based on laboratory LC50 and
point 60-80 曟 for h[33]. The extracts were filtered through a LC90 values) quantity for each treatment was determined by
Buchner funnel with Whatman number 1 filter paper. The calculating the total surface area of drinking water bodies
crude plant extracts were evaporated to dryness in rotary in each habitat. The required quantities of E. hirta and Bs
vacuum evaporator. One gram of the plant residue was were mixed thoroughly with water in a bucket with constant
dissolved in 100 mL of acetone (stock solution) considered agitation. Teepol was used as emulsifying agent (0.05%).
as 1% stock solution. From this stock solution concentrations Field applications of the E. hirta leaf extracts and Bs were
were prepared ranging from75, 150, 225, 300 and 375 ppm, done with the help of a knapsack sprayer (Sujatha Products,
respectively. India, Private Limited, 2010) and uniformly on the surface of
the drinking water bodies in each habitat. Dipper sampling
2.5. Microbial bioassay and counting of larvae monitored the larval density before
24, 48 and 72 h after the treatment. A separate sample was
B. sphaericus was obtained from T- Stanes & Company taken to determine the composition of each larval habitat.
Limited, Research and Development Coimbatore, Tamil Six trails were conducted for E. hirta of the plant extracts
Nadu, India. The organism was grown in a liquid medium and B. sphaericus alone and combined the treatment.
containing (in grams per liter of distilled water): FeSO4·7H2O, The percentage of reduction was calculated by the
0.01; MnSO4, 0.1; MgSO4·7H2O, 0.2; CaCl2, 0.08; K2HPO4, 0.025; following formula:
yeast extract, 2; peptone, 4; and D-glucose, 1 and casein,
C-T
5. Solutions of yeast extract, peptone casein, D-glucose, Percentage of reduction = 伊100
K 2HPO 4 and C a C l 2 were separately prepared, sterilized, C
and added before inoculation. The pH of the medium was Where C is the total number of mosquitoes in control, T is
adjusted to 7.1 before sterilization. The required quantity of the total number of mosquitoes in treatment.
B. sphaericus was thoroughly mixed with distilled water and
prepared at various concentrations ranging from 10, 20, 40, 2.8. Statistical analysis
60 and 80 ppm, respectively.
All data were subjected to analysis of variance; the means
2.6. Larval/pupal toxicity test were separated using Duncan's multiple range tests by Alder
et al[36]. The average larval mortality data were subjected to
Laboratory colonies of mosquito larvae/pupae were used probit analysis for calculating, LC50, LC90 and other statistics
for the larvicidal/pupicidal activity. Twenty-five numbers of at 95% confidence limits of upper confidence limit (UCL)
first to fourth instars larvae and pupae were introduce into and lower confidence limit (LCL) and chi-square values
500 mL glass beaker containing 249 mL of de-chlorinated calculated using the SPSS 16.0 version (Statistical software
water and 1 mL of desired concentrations of plant extract package). The values were expressed as mean±standard
4 K. Kovendan et al./Asian Pacific Journal of Tropical Medicine (2013)412-420

deviation of five replicates. Results with P< 0 . 05 were ppm, respectively. The LC50 value of pupae was 332.39 ppm,
considered to be statistically significant. and the LC90 value of pupae was 779.80 ppm, respectively.
Table 2 shows the larval mortality of An. stephensi (Ⅰ to
Ⅳ instars) after the treatment of B. sphaericus at different
3. Results concentrations (10 to 80 ppm). Thirty two percent mortality
was noted at 1 st instar larvae by the treatment of B.
Larval and pupal mortality of An. stephensi after the sphaericus at 10 ppm, whereas it has been increased to 69.4%
treatment of methanol extract of E. hirta leaf extract was at 80 ppm of B. sphaericus treatment. Similar trends have
observed. Table 1 illustrates the larval and pupal mortality been noted for all the instars of An. stephensi at different
of An. stephensi (Ⅰ to Ⅳ instars) after the treatment of E. concentrations. The LC50 and LC90 values were represented
hirta at different concentrations (75 to 375 ppm). 40.8% as follows: the LC50 value of 1st instar was 44.29 ppm, 2nd
mortality was noted at 1st instar larvae by the treatment of instar was 55.83 ppm, 3rd instar was 68.51 ppm, and 4th
E. hirta at 75 ppm, whereas it has been increased to 81.6% instar was 82.19 ppm, and pupa was 95.55 ppm, respectively.
at 375 ppm of E. hirta leaf extract treatment. Similar trend The LC90 value of 1st instar was 138.27 ppm, 2nd instar was
has been noted for all the instars of An. stephensi at different 165.17 ppm, 3rd instar was 178.30 ppm, and 4th instar was
concentration of E. hirta treatment. The LC50 and LC90 values 199.17 ppm, and pupa was 213.06 ppm, respectively.
were represented as follows: LC50 value of 1st instar was Table 3 provides the combined larval mortality after
137.40 ppm, 2nd instar was 172.65 ppm, 3rd instar was 217.81 treatment of EHLE and B. sphaericus for all the larval
ppm, and 4th instar was 269.37 ppm, respectively. The LC90 instars. The concentration at 75+40 combined EHLE + B.
value of 1st instar was 470.69 ppm, 2nd instar was 531.43 sphaericus treatment for 4th instar larval mortality was 69.2%.
ppm, 3rd instar was 590.77 ppm, and 4th instar was 685.60 LC50 and LC90 values were represented as follows: LC50 value

Table 1
Larval and pupal toxicity effect of E. hirta against malarial vector, An. stephensi.
Mosquito larval instars % of Larval and pupal mortality 依 SD
LC50 (LCL- UCL) LC90 (LCL - UCL) χ2(df=4)
and pupa 75 ppm 150 ppm 225 ppm 300 ppm 375 ppm
1st instar 40.8依0.7 51.2依1.3 63.4依1.0 74.0依2.0 81.6依1.8 137.40(90.57-170.06) 470.69(407.32-581.00) 0.05*
2nd instar 36.0依1.4 47.2依1.3 58.4依1.0 65.8依1.7 77.2依0.7 172.65(130.92-204.72) 531.43(453.33-673.66) 0.21*
3 d instar 31.2依1.1 40.4依1.3 52.8依1.7 58.6依1.4 71.6依1.6 217.81(182.54-251.67) 590.77(498.56-763.33) 0.45*
r

4th instar 27.2依1.7 36.0依1.4 45.6依1.8 51.8依1.6 63.6依2.0 269.37(232.85-317.28) 685.60(563.16-934.94) 0.23*
Pupa 22.6依1.3 30.4依1.0 39.0依1.4 45.0依0.6 55.2依0.7 332.39(287.54-410.88) 779.80(625.52-1117.81) 0.13*
Control-Nil mortality, LCL - Lower confidence Limit, UCL - Upper confidence Limit, χ - Chi-square value, df - degrees of freedom.
2

*Significant at P < 0.05 level.

Table 2
Larval and pupal toxicity effect of B. sphaericus against malarial vector, An. stephensi.
Mosquito larval instars % of Larval and pupal mortality 依 SD
and pupa LC50 (LCL- UCL) LC90 (LCL - UCL) χ2(df=4)
10 ppm 20 ppm 40 ppm 60 ppm 80 ppm
1st Instar 32.0依1.2 38.2依1.6 46.0依1.4 58.2依0.9 69.4依1.0 44.29(35.82-53.33) 138.27(113.53-185.90) 0.19*
2nd Instar 29.2依0.7 35.0依0.6 41.0依1.4 52.6依1.8 61.2依1.7 55.83(46.17-69.82) 165.17(130.71-240.01) 0.20*
3rd Instar 25.0依1.4 29.2依1.1 36.4依1.0 44.2依1.3 56.8依1.7 68.51(57.42-88.10) 178.30(140.17-262.35) 0.26*
4th Instar 21.8依1.1 26.4依1.0 31.2依0.7 36.0依1.4 52.4依1.8 82.19(67.83-112.08) 199.17(153.10-308.48) 1.44*
Pupa 18.2依1.1 21.6依0.8 26.0依1.4 31.4依1.0 46.2依0.7 95.55(77.86-135.38) 213.06(162.21-337.40) 1.07*
Control-Nil mortality, LCL - Lower confidence Limit, UCL - Upper confidence Limit, χ2 - Chi-square value, df - degrees of freedom.
*Significant at P < 0.05 level.

Table 3
Combined effect of larval and pupal activity of methanol extract of E. hirta and B. sphaericus against malarial vector, An. stephensi.
Mosquito larval instars % of Larval and pupal mortality 依 SD χ2(df=4)
and pupa LC50 (LCL- UCL) LC90 (LCL - UCL)
75 +5 75 + 10 75 + 20 75 +30 75 + 40
1st Instar 52.4依1.8 66.8依1.9 72.2依1.6 88.6依1.3 99.6依0.4 79.13(59.07-86.14) 104.26(96.92-126.35) 7.15*
2nd Instar 44.2依1.7 63.8依1.9 67.4依1.3 83.0依1.4 88.4依1.3 80.42(74.14-84.55) 115.91(110.24-125.23) 3.85*
3rd Instar 39.0依1.4 53.6依1.9 60.4依1.0 69.8依0.7 77.0依1.6 86.01(79.17- 90.51) 133.75(123.32- 154.12) 1.85*
4th Instar 31.2依2.3 46.6依1.0 56.2依0.7 61.0依1.4 69.2依1.1 93.00(87.71- 97.50) 144.02(130.85- 170.95) 3.28*
Pupa 28.4依1.3 39.6依1.0 52.6依1.6 56.2依1.4 63.4依1.8 98.12(93.37- 103.50) 151.34(136.01- 183.77) 2.69*

Control-Nil mortality, LCL - Lower confidence Limit, UCL - Upper confidence Limit, χ - Chi-square value, df - degrees of freedom
2

*Significant at P < 0.05 level.

 K. Kovendan et al./Asian Pacific Journal of Tropical Medicine (2013)412-420
5

of 1st instar was 79.13%, 2nd instar was 80.42%, 3rd instar year, leaving as many as 2 100 million people at risk around
was 86.01% and 4th instar was 93.00%, respectively. The LC90 the world[38]. The secondary metabolite of plant origins
value of 1st instar was 104.26 ppm, II instar was 115.91 ppm, makes up a vast repository compounds with a wide range
III instar was 133.75 ppm, and IV instar was 144.02 ppm, of biological activities. There have been many reports of
respectively. The χ2 values are significant at P<0.05 level. higher plant extracts possessing relatively good potential
The 95% confidence limits LC50, LC90 (LCL-UCL) values were to inhibit viruses[39]. The methanol extract of Spheranthus
also calculated. Larval and pupal mortality was observed indicus showed macro filaricidal activity by worm motility
after 24 h exposure. No mortality was observed in the control and subsequent mortality was observed [40]. The latex of
group. Calotropis procera has shown larvicidal efficacy against all
Total number 425 An. stephensi larvae found were observed three important vector species, Aedes aegypti, An. stephensi
in the drinking water body systems. After treated with E. and Culex quinquefasciatus in India[41].
hirta against An. stephensi larval density was reduced by The direct and indirect contributions of such effects to
13.17%, 37.64% and 84.00% at 24, 48 and 72 h, respectively. treatment efficacy through reduced larval feeding and fitness
Similarly, the reductions of An. stephensi larval densities need to be properly understood in order to improve the use
after treatment with B. sphaericus were 8.47%, 29.41% and of botanical insecticides for of An. stephensi. These and other
79.52%, respectively. Combined effect of E. hirta and B. naturally occurring insecticides may play a more prominent
sphaericus were 44.23%, 81.64% and 100.0% at 24, 48 and 72 role in mosquito control programs in the future[42]. David et
h, respectively (Table 4, 5). al found that phytochemicals primarily affect the midgut
epithelium and secondarily affect the gastric caeca and the
Table 4 malpighian tubules in mosquito larvae[43]. Furthermore,
Field trail by using plant extracts of E. hirta and B. sphaericus the crude extracts may be more effective compared to the
drinking water tanks against An. stephensi. individual active compounds, due to natural synergism
After treatment that discourages the development of resistance in the
Sample Before
E. hirta B. sphaericus vectors[44]. The leaf methanol extract of Cassia fistula was
No. treatment
24 h 48 h 72 h 24 h 48 h 72 h
tested for larvicidal and ovicidal activity of against Culex
1 85 66 44 15 79 52 20
quinquefasciatus and An. stephensi, with the LC50 values of
2 75 69 59 13 68 72 18
17.97 and 20.57 mg/L, respectively[45].
3 49 37 29 7 44 34 9
4 66 59 48 12 58 52 20
The crude and column chromatographic fractions of the
5 96 89 44 11 90 47 16
methanol leaf extract of Jatropha curcas were tested for
6 54 49 41 10 50 43 14 their larvicidal activities against the laboratory-reared late
Average 70.8 61.5 44.1 11.3 64.8 50 14.5 third instar larvae of Anopheles arabiensis [46]. Crude extract
Total 425 369 265 68 389 300 87 of flower, leaf, and stem of Spilanthes acmella L. plants
were tried against An. stephensi Liston and found that the
Table 5 LC50 and LC90 values of flower extract were more than the
Field trail by using combined effect of drinking water tanks 0.5伊0.5伊 leaf and stem extracts against An. stephensi[47]. The crude
1.0 against An. stephensi. petroleum ether leaf extract of Jatropha curcas to have
Sample No. Before treatment After treatment larvicidal activity with the LC50 of <100 ppm on the early
24 h 48 h 72 h fourth instar larvae of vector mosquitoes including Culex
1 85 55 12 - quinquefasciatus, An. stephensi, and Aedes aegypti [48].
2 75 42 11 - Larvicidal activities of ethanol extract of Allium
3 49 28 9 - sativum (garlic bulb) against the filarial vector, Culex
4 66 30 15 - quinquefasciatus with the LC50 values for the second, third
5 96 51 22 - and fourth larval instars were 144.54, 165.70 and 184.18
6 54 31 9 - ppm, respectively. The results obtained show that this
Average 70.8 39.5 13 0 plant material exhibited significant activity and could be
Total 425 237 78 0 considered as potent natural larvicidal agent [49]. Lippia
citriodora essential oil exhibited an LC50 value of 101.4
ppm against the third instars larvae of An. stephensi[50]. The
4. Discussion larvicidal and pupicidal efficacy of Solanum xanthocarpum
leaf extract with LC50 value of first to fourth instars larvae
Malaria is one of the most common vector-borne diseases and pupae 155.29, 198.32, 271.12, 377.44 and 448.41 ppm,
widespread in tropical and subtropical regions, including respectively. The LC90 value of first to fourth instars larvae
parts of the America, Asia, and Africa[37]. Malaria is the and pupae 687.14, 913.10, 1011.89, 1058.85 and 1 141.65
world's most dreadful tropical disease. Mosquito-borne ppm, respectively[51]. In the present results, E. hirta leaf
diseases are endemic in more than over 100 countries, extract against first to fourth instars larvae and pupae of
causing mortality of nearly two million people every year, An. stephensi has been studied in the laboratory condition.
and at least one million children die of such diseases each The lethal concentrations (LC50/LC90) of E. hirta were 137.40,
6 K. Kovendan et al./Asian Pacific Journal of Tropical Medicine (2013)412-420

172.65, 217.81, 269.37 and 332.39 ppm, respectively. The of neem products were as effective as good quality
LC90 values of 470.69, 531.43, 590.77, 685.60 and 779.80 ppm, crude neem products in the control of culicine vectors of
respectively. Japanese encephalitis and produced a slight but significant
The methanol leaf extract of Calotropis gigantea and reduction in population of anopheline pupae[64]. Azadirachta
bacterial insecticide, B. thuringiensis have mosquitocidal excels Jack showed excellent larvicidal properties at low
property was evaluated as target species of mosquito concentrations against Culex pipiens molestus. Its LC 50
vectors[52]. This is an ideal eco-friendly approach for the value after 1 day was 62.5 毺g/mL[65]. In our recent study,
control of vector control programs. In the present study, B. the field trials were conducted by using Clerodendrum
sphaericus at different concentrations brought out toxicity inerme and Acanthus ilicifolius treatment in different
against the various larval instars malarial vector, An. habitats of three species of mosquito vectors namely An.
stephensi. Similarly, the microbial pesticide spinosad against stephensi, Aedes aegypti, Culex quinquefasciatus (Vadavalli,
the malarial vector, An. stephensi showed 85% mortality. Mettupalayam, Navavoor privu, Pommanampalayam,
The observed mortality rate suggests that the above extract Mettupalayam, Kallaru Ooty) in Tamil Nadu, India. The
can be used as bio-pesticides. The LC50 of second, third percentage reduction of larval mortality also showed the
and fourth instars larvae of An. stephensi were 0.27%, 0.28% variations among the different breeding habitats of mosquito
and 0.30%, respectively[53]. Earlier, ten microbial products vectors at 24, 48 and 72 h. This may be due to the impact of
to develop a strategy to control mosquito larval and pupal geographical distribution of An. stephensi, Aedes aegypti and
population in the lab and field. Highest larval mortality was Culex quinquefasciatus at the breeding sites[66]. Similarly, in
evident in the lab with LC50 and LC90 at 0.25 and 0.5 at 24 h the present study the combined activity of plant extract of E.
for Aedes aegypti[54]. hirta and B. sphaericus in the field were 44.23%, 81.64% and
The toxicity of the wild-type B. thuringiensis subsp. 100.00% at 24, 48 and 72 h, respectively. This results shows
Bacillus israelensis-H14 (Bti) and B. sphaericus-2362 (Bs) that B. sphaericus and EHLE pesticides can control the
was determined towards Aedes aegypti larvae, theLC50 were malarial vector, An. stephensi.
estimated to be 0.094 and 1.18 毺g/mL and LC90 to be 0.179 The current investigation revealed that the crude extract of
and 2.12 毺g/mL, respectively and the Bti and Bs spore- E. hirta and B. sphaericus possesses remarkable mosquito
crystal toxins were assayed in six different proportions properties against An. stephensi mosquitoes. This study
that resulted in LC 50 and LC 90 varying from 0 . 018 to is the first to report on the mosquito combined larvicidal
1.51 毺g/mL and 0.090 to 2.88 毺g/mL, respectively[55]. and pupicidal activity E. hirta and B. sphaericus. These
Strains of B. sphaericus are known to have high activity results show that these two biological agents could reduce
towards larvae of Culex, variable toxicity to Anopheles the malarial incidence. Further studies are in progress
depending on the species, and are inactive against Aedes to evaluate the effect of purified extract on larvicidal
larvae[56]. The larvicidal efficacy of SPH-88 against larvae of activity. The result shows that good larvicidal and pupicidal
Culex quinquefasciatus is higher than that of the same larval properties of against vector control programs.
stages of Anopheles arabiensis[57]. The formulated product
of B. sphaericus BSN-0011 is effective against laboratory
reared Culex quinquefasciatus. In our study the efficacy of Conflict of interest statement
B. sphaericus exhibited significantly higher toxicity on first
instars than on the second instars larvae of An. stephensi[58]. We declare that we have no conflict of interest.
Field trials on the efficacy of mosquito nets treated
with a tablet formulation of deltamethrin (K-OTAB襆) in
Sundargarh District of Orissa state, India showed reduction Acknowledgments
in malaria incidence[59,60]. Efficacy of aqueous suspension
of Bti (Vectobac 12 AS) was investigated in a laboratory The authors are thankful to Defence Research &
and field conditions against Anopheles culicifacies and An. Development Organisation-Life Sciences Research Board,
stephensi and found effective[61] Field trials were conducted Ministry of Defence, Govt. of India, New Delhi, for providing
at Anwona and Mmemiriwa villages located at Ghana on financial support for the present work. The authors are
residual activity of deltamethrin-impregnated durable grateful to Mr. N. Muthukrishnan, Technician and Mr. A.
residual wall lining against susceptible Anopheles gambiae Anbarasan, Lab Assistant, National Centre for Diseases
even 3 weeks after installation on both cement and mud Control, Mettupalayam, Tamil Nadu for their helping
surfaces, found 100% mortality on both surfaces using WHO mosquito collection and mosquito samples provided for the
cone bioassay kits[62]. More field studies are needed to present work.
establish the utility of these interventions at personal and
household levels. Larvicidal activity of the emulsified neem
oil formulation was observed against late instars of An. References
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