Asian Pacific Journal of Tropical Medicine (2013)847-853 847

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Asian Pacific Journal of Tropical Medicine

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Document heading doi:

Larvicidal efficacy of Catharanthus roseus Linn. (Family: Apocynaceae)

leaf extract and bacterial insecticide Bacillus thuringiensis against Anopheles
stephensi Liston.
Chellasamy Panneerselvam1,2, Kadarkarai Murugan2, Kalimuthu Kovendan2*, Palanisamy Mahesh
Kumar2, Sekar Ponarulselvam2, Duraisamy Amerasan2, Jayapal Subramaniam2, Jiang-Shiou Hwang3
1
DRDO-BU Center for Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
2
Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
3
Institute of Marine Biology, National Taiwan Ocean University, Keelung 202-24, Taiwan

ARTICLE INFO ABSTRACT

Article history: Objective: To explore the larvicidal activity of Catharanthus roseus (C. roseus) leaf extract and
Received 10 August 2013
Bacillus thuringiensis (B. thuringiensis) against the malarial vector Anopheles stephensi (An.
Received in revised form 15 September 2013
Accepted 15 October 2013
stephensi), when being used alone or together. Methods: The larvicidal activity was assayed at
Available online 20 November 2013 various concentrations under the laboratory and field conditions. The LC50 and LC90 values of
the C. roseus leaf extract were 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
Keywords: petroleum ether extract of C. roseus against the first to fourth instars larvae with LC50=3.34, 4.48,
Catharanthus roseus 5.90 and 8.17 g/L, respectively; B. thuringiensis against the first to fourth instars larvae with
Bacillus thuringiensis LC50=1.72, 1.93, 2.17 and 2.42 g/L, respectively; and the combined treatment with LC50=2.18, 2.41,
Anopheles stephensi 2.76 and 3.22 g/L, respectively. No mortality was observed in the control. Conclusions: The
Larvicidal activity petroleum ether extract of C. roseus extract and B. thuringiensis have potential to be used as ideal
eco-friendly agents for the control of An. stephensi in vector control programs. The combined
treatment with this plant crude extract and bacterial toxin has better larvicidal efficacy against
An. stephensi.

In India, malaria is transmitted by six vector species, in

1. Introduction which Anopheles stephensi (An. stephensi) is responsible in
urban areas[4]. Mosquitoes in the larval stage are attractive
Mosquitoes are the principal vector of many vector-borne targets for pesticides because they breed in water; thus,
diseases affecting human beings and animals, in addition it is easy to deal with them in this habitat. Management
to nuisance. Vector-borne diseases in India, e.g., malaria, of disease vector using synthetic chemicals has failed
dengue, chikungunya, filariasis, Japanese encephalitis, and because of resistance, effect on non-target organisms and
leishmaniasis, cause thousands of deaths per year. India environmental pollution. On the other hand, the recent
reports 1.48 million malarial cases and about 1 173 deaths, public perception against the vector control using synthetic
1.4 million suspected and 1 985 confirmed chikungunya chemicals has shifted the research effort towards the
cases, 5 000 Japanese encephalitis cases and approximately development of environmentally sound and biodegradable
1 000 deaths, and 383 dengue cases and 6 deaths during agents. In that way, plant extracts including essential
2006 and 2007[1-3]. oils have attracted much attention to control the vector
*Corresponding author: Dr. K. Kovendan, Division of Entomology, Department of
transmitted diseases[5].
Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641046, India. Plants are rich sources of bioactive compounds that can
Tel: +91-9962447932
E-mail: [email protected] be used to develop environmentally safe vector and pest-
848 Chellasamy Panneerselvam et al./Asian Pacific Journal of Tropical Medicine (2013)847-853

managing agents. A number of plants and microbes have a variety of habitats[25,26]. In this context, the present study
been reported as selectable subjects with little or no harmful was designed to evaluate the mosquito larvicidal effects of
effect on non-target organisms and the environment[6,7]. C. roseus and B. thuringiensis against mosquito larvae An.
Botanical phytochemicals with mosquitocidal potential are stephensi under laboratory as well as field conditions.
now recognized as potent alternative insecticides to replace
synthetic insecticides in mosquito control programs due
to their excellent larvicidal, pupicidal, and adulticidal 2. Materials and methods
properties. Many researchers have reported the effectiveness
of plant extract against mosquito larvae[8-10]. 2.1. Collection of eggs and maintenance of larvae
Madagascar Periwinkle [Catharanthus roseus (C. roseus)]
belonging to the Apocynaceae family is formerly known as The eggs of An. stephensi were collected from National
Vinca rosea. It is one of the important medicinal plants, due Centre for Disease Control field station of Mettupalayam,
to the presence of the indispensable anti-cancer drugs, Tamil Nadu, India, using an “O”-type brush. These eggs
vincristine and vinblastine. It can erect bushy perennial were brought to the laboratory and transferred to 18 cm 伊
herb and evergreen shrub grows to a height of 90 cm with 13 cm 伊 4 cm enamel trays containing 500 mL of water for
a spread of 1 m. Its leaves are simple, opposite, exstipulate hatching. The mosquito larvae were fed on pedigree dog
and petiolate. It contains more than 70 alkaloids mostly biscuits and yeast at a mass ratio of 3:1. The feeding was
of the indole type. It has medicinal importance owing to continued until the larvae transformed into the pupal stage.
the presence of alkaloids like ajamalicine, serpentine and
reserpine, which are well known for their hypotensive and 2.2. Maintenance of pupae and adults
antispasmodic properties. The root bark contains alkaloid
alstonine which has been used traditionally for its calming T he pupae were collected from the culture trays and
effect and its ability to reduce blood pressure. C. roseus transferred to plastic containers (12 cm 伊 12 cm) containing
exhibited high in vitro anti-plasmodial activity, which may 500 mL of water with the help of a dipper. The plastic jars
be due to the presence of compounds such as alkaloids, were kept in a 90 cm 伊 90 cm 伊 90 cm mosquito cage for
terpenoids[11], flavonoids[12] and esquiterpenes[13] that were adult emergence. Mosquito larvae were reared at (27依2) 曟
previously separated from the plant. with 75%-85% relative humidity under a photoperiod of 14
Bacillus thuringiensis ( B. thuringiensis ) subsp. var h/10 h (light/dark). A 100 g/L sugar solution was provided for
israelensis ( B ti ) is a G ram positive bacterium able to a period of 3 d before blood feeding.
synthesize endotoxin protein crystals during sporulation. Bti
produces four major insecticidal cryptochrome proteins and 2.3. Blood feeding of adult An. stephensi
three cytolytic proteins[14]. The ingestion of these crystals
by mosquito larvae rapidly leads to the formation of pores, The adult female mosquitoes were allowed to feed on the
cell lysis, septicemia and finally larva death[15,16]. One of blood of a rabbit (a rabbit per day, exposed on the dorsal
the main advantages of Bti toxins is their capacity to act side) for 2 d to ensure adequate blood feeding for 5 d. After
synergistically, improving the toxicity of the mixture[17,18] and blood feeding, enamel trays with water from the culture trays
reducing resistance to cryptochrome toxins in mosquitoes[19]. were placed in the cage as oviposition substrates.
B iological control is an important component of the
integrated vector control strategy and is being practiced in 2.4. Collection of plant and preparation of extract
many countries for the control of mosquitoes[20,21].
Bacillus sp. produces large, spreading, gray-white C. roseus plants were collected in and around Maruthmalai
colonies with irregular margins. A unique characteristic of hills, C oimbatore, I ndia. T he plants were identified at
this bacterium is its ability to produce endospores when Botanical Survey of India, Coimbatore, India. C. roseus
environmental conditions are stressful. B. thuringiensis leaves were washed with tap water and dried in shade at
is a plant growth promoting bacterium which produces room temperature. The dried plant materials (leaves) were
bacteriocin compounds of insecticidal properties and is powdered by an electrical blender. From the powder, 500 g
marketed worldwide for control of many important plant of the plant material were extracted with 1.5 L of organic
pests, mainly caterpillars of Lepidoptera, mosquito larvae solvents of petroleum ether using a Soxhlet apparatus at a
and black flies[22]. Well-known bacterial agents which boiling point of 60-80 曟 for 8 h[27]. The extracts were filtered
have been used successfully for mosquito control are B. through a Buchner funnel with Whatman number 1 filter
thuringiensis and Bacillus sphaericus (B. sphaericus)[23,24]. paper. The crude plant extracts were evaporated to dryness
Two bacterial agents, B. thuringiensis and B. sphaericus, in a rotary vacuum evaporator. Twenty gram of the plant
are being widely used for control of mosquito breeding in residue was dissolved in 100 mL of acetone (stock solution)
 Chellasamy Panneerselvam et al./Asian Pacific Journal of Tropical Medicine (2013)857-853
849

considered as 200 g/ L stock solution. F rom this stock were conducted for the C. roseus extract and B. thuringiensis
solution, concentrations of 20, 50, 80, 110 and 140 g/L were alone and for the combined treatment, respectively. The
prepared, respectively. percentage of reduction was calculated using the following
formula:
2.5. Microbial bioassay Percentage of reduction=(C-T)/C×100%. (2)
where, C is the total number of mosquitoes in control, and
B. thuringiensis “subsp” was obtained from Tuticorin T is the total number of mosquitoes in treatment.
Alkali Chemicals and Fertilizers Limited, Chennai, India.
B. thuringiensis 630 ITU/mg (a.i.), 5.0% (w/w); total proteins 2.8. Statistical analysis
[including the active ingredient 5.0% (w/w)], 10.0% (w/w);
fermentation solids, 10.0% (w/w); inert ingredient, 48.0% (w/ All data were subjected to analysis of variance, and means
w); non-ionic surfactant, 0.2 (w/w); food grade preservative, were separated using D uncan’s multiple range test[30].
0.3%; UV protectant, 0.1%; and water, 71.4% were used. Total The average larval mortality data were subjected to probit
100.0% (w/w) was active specifically against mosquito larvae. analysis for calculating LC50, LC90 and other statistics at 95%
The required quantity of B. thuringiensis was thoroughly confidence limits of upper fiducidal limit (UFL) and lower
mixed with distilled water and prepared to various fiducidal limit (LFL), and chi-square values were calculated
concentrations, ranging from 10 to 30 g/L, respectively. using the SPSS 16.0 version (software package). The values
are expressed as mean依SD of five replicates. Results with
2.6. Larval toxicity test P<0.05 were considered to be statistically significant.

Laboratory colonies of mosquito larvae were used for

the larvicidal activity. The first to fourth instar larvae and 3. Results
pupae, 25 each, were respectively introduced into a 500-
mL glass beaker containing 249 mL of de-chlorinated water 3.1. Larval toxicity of the C. roseus extract against An.
and 1 mL of desired concentrations of plant extract and stephensi under laboratory conditions
B. thuringiensis. In fact, 0.5 mg larval food was provided
for each test concentration. At each tested concentration, The larval mortality of An. stephensi after the treatment
two to five trials were made and each trial consisted of five with the petroleum ether extract of C. roseus leaves was
replicates. The control was set up by mixing 1 mL of acetone observed. Table 1 provides the larval mortality of An. stephensi
with 249 mL of dechlorinated water. The larvae exposed to ( the first to fourth instars ) after the treatment with the
de-chlorinated water without acetone served as control. The C. roseus extract at various concentrations. A mortality of
mortalities (%) were corrected by using the following Abbott’s 47% was noted in the first instar larvae with the treatment of
formula[28], and lethal concentrations LC50 and LC90 were C. roseus at 20 g/L, and it gradually increased to 94% when
calculated from toxicity data by using probit analysis[29]. the C. roseus leaf extract was used at 14 g/L.
C orrected mortality= ( O bserved mortality in treatment- Similar increasing trend was noted for all the instars of
Observed mortality in control)/(100%-control mortality)×100%.(1) An. stephensi when treated with the C. roseus extract at
different concentrations. The LC50 and LC90 values of the
2.7. Field trial C. roseus extract alone against the An. stephensi larvae are
also represented in Table 1.
For the field trial, the quantity of plant extract residues and
required quantity of Bti (based on laboratory LC50 and LC90 3.2. Larval toxicity of B. thuringiensis against An. stephensi
values) for each treatment were determined by calculating under laboratory conditions
the total surface area of sewage water bodies in each habitat.
The required quantities of C. roseus and Bti were mixed Table 2 illustrates the larval mortality of An. stephensi
thoroughly with water in a bucket with constant agitation. ( the first to fourth instars ) after the treatment with
Teepol was used as emulsifying agent (0.05%, w/w). Field B. thuringiensis at different concentrations. A mortality of
applications of the C. roseus leaf extracts and Bti were done 33% was noted in the first instar larvae after the treatment
with the help of a knapsack sprayer (Sujatha Products, India, with B. thuringiensis at 10 g/L, and it increased to 84% at
2010) and uniformly sprayed on the surface of the sewage 30 g/ L . S imilar increasing trend was noted for all the
water bodies in each habitat. Dipper sampling and counting instars of An. stephensi when treated with B. thuringiensis
of larvae were done to monitor the larval density after 24, 48, at different concentrations. The LC50 and LC90 values of
and 72 h post the treatment. A separate sample was taken to B. thuringiensis alone against the An. stephensi larvae are
determine the composition of each larval habitat. Six trials also represented in Table 2.
850 Chellasamy Panneerselvam et al./Asian Pacific Journal of Tropical Medicine (2013)847-853

after the treatment with the C. roseus extract alone, the larval
3.3. Larval toxicity of the combined C. roseus leaf extract density of An. stephensi was reduced by 12.26%, 28.80% and
and B. thuringiensis against An. stephensi under laboratory 79.46% at 24, 48, and 72 h, respectively. Similarly, the larval
conditions density was reduced by 10.93%, 25.86%, and 75.73% after 24,
48, and 72 h post the treatment with B. thuringiensis alone,
Table 3 shows the considerable larval mortality of all the respectively. The combined application of the C. roseus
larval instars after the combined treatment with C. roseus extract and B. thuringiensis caused 20.53%, 77.33%, and
leaf extract and B. thuringiensis. After treatment with the 100.00% reduction of larval density after 24, 48, and 72 h,
C. roseus extract at 60 g/L and B. thuringiensis at 2.5 g/L, respectively. The laval density after treatment is shown in
the mortality of the An. stephensi in each larval stage was Table 4.
highest. The LC50 and LC90 values of the combined C. roseus
leaf extract and B. thuringiensis against the An. stephensi
larvae are also represented in Table 3. 4. Discussion

3.4. Larval toxicity of the combined C. roseus leaf extract and Malaria is the largest single component of disease burden;
B. thuringiensis against An. stephensi under field conditions epidemic malaria, in particular, remains a major public
health concern in tropical countries. In many developing
A total number of 375 An. stephensi larvae were observed in countries, especially in Africa, malaria exacts an enormous
the overhead tanks of water body systems. In the field trial, toll in lives, medical costs, and days of labor lost[31].

Table 1
Larval toxicity of C. roseus leaf extract against An. stephensi.
Larval mortality (%) at different concentrations of extract
Larval instar LC50 (LFL-UFL) (g/L) LC90 (LFL-UFL) (g/L) 氈2 (df=4)
20 g/L 50 g/L 80 g/L 110 g/L 140 g/L
First instar 47.00依1.78 58.00依1.41 66.00依1.26 79.00依0.89 94.00依1.49
e e d f d
3.34 (1.62-4.54) 14.08 (12.40-16.70) 4.21*
Second instar 42.00依1.63d 54.00依1.09d 60.00依1.41c 68.00依1.32cd 86.00依1.16c 4.48 (2.56-5.81) 18.07 (15.44-22.72) 3.24*
Third instar 38.00依1.35bc 48.00依1.41bc 54.00依1.85b 66.00依1.01c 77.00依1.93b 5.90 (4.11-7.24) 21.06 (17.63-27.54) 0.62*
Fourth instar 29.00依1.41a 37.00依1.85a 49.00依1.72a 62.00依1.62ab 70.00依1.16ab 8.17 (6.92-9.45) 21.90 (18.60-27.79) 0.24*
The larval mortalities are expressed as mean依SD of five replicates. Nil mortality was observed in the control. Within a column, means followed
by the same letter(s) are not significantly different at 5% level by Duncan’s multiple range test. LFL, lower fiducidal limit; UFL, upper fiducidal
limit; df, degrees of freedom; 氈2, chi-square value. *Significant at P<0.05 level.

Table 2
Larval toxicity of B. thuringiensis against An. stephensi.
Larval mortality (%) at different concentrations of B. thuringiensis
Larval instar LC50 (LFL-UFL) (g/L) LC90 (LFL-UFL) (g/L) 氈2 (df=4)
10 g/L 15 g/L 20 g/L 25 g/L 30 g/L
First instar 33.00依1.32d 41.00依1.72d 59.00依1.41d 67.00依1.16d 84.00依1.85e 1.72 (1.52-1.88) 3.55 (3.21-4.10) 1.73*
Second instar 29.00依1.85c 36.00依1.41c 52.00依1.93c 62.00依1.72bc 78.00依1.16d 1.93 (1.75-2.11) 3.87 (3.46-4.54) 1.06*
Third instar 26.00依1.72b 32.00依1.16b 47.00依1.41b 55.00依0.80b 71.00依1.32c 2.17 (1.98-2.38) 4.31 (3.79-5.21) 0.99*
Fourth instar 22.00依1.32ab 29.00依1.6a 40.00依1.72a 51.00依1.85a 64.00依1.41ab 2.42 (2.21-2.69) 4.67 (4.05-5.76) 0.20*
The larval mortalities are expressed as mean依SD of five replicates. Nil mortality was observed in the control. Within a column, means followed
by the same letter(s) are not significantly different at 5% level by Duncan’s multiple range test. LFL, lower fiducidal limit; UFL, upper fiducidal
limit; df, degrees of freedom; 氈2, chi-square value. *Significant at P<0.05 level.

Table 3
Larval and pupal toxicity of combined C. roseus leaf extract and B. thuringiensis against An. stephensi.
Larval mortality (%)
Larval instar LC50 (LFL-UFL) (g/L) LC90 (LFL-UFL) (g/L) 氈2 (df=4)
20/0.5 30/1.0 40/1.5 50/2.0 60/2.5
† † † † †

First instar 52.00依1.85d 71.00依1.41 80.00依1.32 89.00依1.16d

d d
97.00依1.72d 2.18 (1.56-2.59) 5.09 (4.71-5.67) 2.30*
Second instar 47.00依1.41c 66.00依1.72c 72.00依0.74c 81.00依1.60c 92.00依1.16c 2.41 (1.70-2.87) 6.08 (5.52-6.99) 3.07*
Third instar 41.00依1.72b 62.00依1.41b 68.00依1.85b 75.00依1.35bc 88.00依1.93b 2.76 (2.10-3.19) 6.73 (6.05-7.87) 4.44*
Fourth instar 35.00依1.85a 56.00依1.62a 61.00依1.41a 74.00依1.72a 82.00依1.16a 3.22 (2.68-3.61) 7.26 (6.50-8.55) 3.86*
indicates the concentrations of the extract (g/L) followed by that of B. thuringiensis (g/L), and the two concentrations are separated by a slash.
†

The larval mortalities are expressed as mean依SD of five replicates. Nil mortality was observed in the control. Within a column, means followed
by the same letter(s) are not significantly different at 5% level by Duncan’s multiple range test. LFL, lower fiducidal limit; UFL, upper fiducidal
limit; df, degrees of freedom; 氈2, chi-square value. *Significant at P<0.05 level.
 Chellasamy Panneerselvam et al./Asian Pacific Journal of Tropical Medicine (2013)857-853
851
Table 4
Larval density in field trial by using leaf extracts of C. roseus and bacterial insecticide B. thuringiensis against malarial vector, An. stephensi
(drinking water tanks with size of 0.10 m 伊 0.10 m 伊 2.00 m).
After treatment with After treatment with
Before After the combined treatment
Sample no. C. roseus extract B. thuringiensis
treatment
24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h
1 44 39 28 12 41 31 14 31 18 0
2 55 48 39 18 49 44 17 39 12 0
3 62 51 43 10 53 49 11 51 8 0
4 71 66 54 12 64 51 18 62 19 0
5 89 76 62 17 78 54 21 73 16 0
6 54 49 41 8 49 49 10 42 12 0
Total 375 329 267 77 334 278 91 298 85 0
Average 62.5 54.8 44.5 12.8 55.6 46.3 15.6 49.6 14.6 0
Reduction percentage (%) - 12.26 28.80 79.46 10.93 25.86 75.73 20.53 77.33 100.00

The larvicidal activity of many plant extracts against kurstaki and botanical insecticides caused a 2 -fold
malarial vectors has been investigated. The petroleum decrease in the gut enzyme activity of larvae of rice
ether ( 60 - 80 曟 ) extracts of Vitex negundo leaves were leaf folder (Cnaphalocrocis medinalis) even at reduced
evaluated for larvicidal activity with LC50 of 2.488 3 mg/L concentrations. A synergistic effect was however found
and LC90 of 5.188 3 mg/L against Culex tritaeniorhynchus[32]; when botanical insecticides and bacterial toxins were
the benzene, petroleum ether, ethyl acetate and methanol combined at low doses. These effects were most obvious
extracts of Citrullus vulgaris leaves were tested for larvicidal in early instars[36]. In another laboratory study, Nathan
activity with LC50 values of 18.56, 48.51, 49.57 and 50.32 mg/ et al reported that the ingestion of bacterial toxins,
L against An. stephensi, respectively[33]; and the compound B. thuringiensis (Berliner) subsp. kurstaki, neem seed kernel
beta-sitosterol isolated from petroleum ether extract of extract and Vitex negundo L. (Lamiales: Verbenaceae) leaf
Abutilon indicum showed LC50 values of 11.49, 3.58 and 26.67 extract by rice leaf folder resulted in an altered leaf-folding
mg/L against Aedes aegypti (Ae. aegypti), An. stephensi and behavior and biology. With the combination of Bti and
Culex quinquefasciatus (Cx. quinquefasciatus), respectively[34]. botanicals, the average leaf consumption was decreased by
In previous study, the oils of 41 plants were evaluated for a factor of 2 even at reduced concentrations when compared
their effects against the third-instar larvae of An. stephensi, with the controls. During larval and pupal stages, adult
Ae. aegypti and Cx. quinquefasciatus. A t first, the oils longevity and fecundity were more affected by the treatments
were surveyed against Ae. aegypti using 50 mg/L solution. with the combination of both bacterial toxins and botanicals
T hirteen oils from 41 plants ( camphor, thyme, amyris, than by the treatment with the bacterial toxins or botanicals
lemon, cedarwood, frankincense, dill, myrtle, juniper, black individually[37]. Well-known bacterial agents which have
pepper, verbena, helichrysum and sandalwood) induced been used successfully for mosquito control are Bti and B.
100% mortality after 24 h or even after shorter periods. The sphaericus[23,24], and Bacillus subtilis produce mosquitocidal
pest oils were tested against the third-instar larvae of the toxins. H owever, they have not been fully studied for
three mosquito species at concentrations of 1, 10, 50, 100 and the nature of their toxins or their biocontrol potential[38].
500 mg/L.The LC50 values of three oils ranged between 1.0 and The lyophilized powder of purified Cyt1A crystals of B.
101.3 mg/L against Ae. aegypti, between 9.7 and 101.4 mg/L thuringiensis was much more toxic and yielded a LC50 of
for An. stephensi, and between 1 . 0 and 50 . 2 mg/ L for 11.332 mg/L[39]. In the present results, the LC50 values of
Cx. quinquefasciatus[5]. B. thuringiensis against the first to fourth instars larvae
Larvicidal activity of Leucas aspera leaf extract against An. were 17.2, 19.3, 21.7, and 24.2 g/L, respectively, and the
stephensi was demonstrated with LC50 values of 96.95 g/L corresponding LC90 values were 35.5, 38.7, 43.1, and 46.7 g/L,
for the first instar, 102 . 72 g/ L for the second instar, respectively.
08.23 g/L for the third instar, 113.03 g/L for the fourth instar S ingh and P rakash have reported that six different
and 127.32 g/L for pupae[35]. In the present results, the C. concentrations of B. sphaericus ( 5 , 10 , 20 , 30 , 40
roseus leaf extract showed considerable mortality against and 50 mg/ L ) were used in laboratory bioassays for
An. stephensi with the LC50 values of 33.4, 44.8, 59.0, and An. stephensi[40]. Similarly, in the case of Cx. quinquefasciatus,
81.7 g/L against the first to fourth instars larvae, respectively, six statistically significant different concentrations of
and the corresponding LC90 values were 140.8, 180.7, 210.6, B. sphaericus were used (0.01, 0.04, 0.05, 0.10, 5.00 and
and 219.0 g/L, respectively. 10 . 00 mg/ L ) . I t was recorded that the mortalities were
Nathan et al reported that combination of B. thuringiensis different for the different instars of Cx. quinquefasciatus and
852 Chellasamy Panneerselvam et al./Asian Pacific Journal of Tropical Medicine (2013)847-853

An. stephensi after exposure of 24 h. Mahesh Kumar et Acknowledgments

al have reported the larvicidal and pupicidal efficacy
of Solanum xanthocarpum against Cx. quinquefasciatus W e thank to D r. K . S asikala, P rofessor and H ead,
with the LC50 value of 155.29, 198.32, 271.12, 377.44 and Department of Zoology, Bharathiar University for providing
448.41 g/L against the first to fourth instars larvae and
the laboratory facilities. T he authors are grateful to
Technician Mr. N. Muthukrishnan and Lab Assistant Mr. A.
pupae, respectively. The LC90 values against the first to
Anbarasan, National Centre for Diseases Control (NCDC),
fourth instars larvae and pupae were 687.14, 913.10, 1 011.89, M ettupalayam, T amil N adu for their helping mosquito
1 058.85, and 1 141.65g/L, respectively[41]. In the present collection and providing mosquito samples for the present
results, the LC 50 values of combined C. roseus and B. work.
thuringiensis against the first to fourth instars larvae
were 21.8, 24.0, 27.5, and 32.1 g/L, respectively, and the
corresponding LC90 values were 50.9, 60.8, 67.3, and 72.6 g/L, References
respectively.
Rao et al reported that the field-tested relatively stable [1] W HO. Combating waterborne diseases at the household level.
Part1: Household water management and health. Geneva: WHO
lipid-rich fractions of neem products were as effective as
Press; 2007.
good quality crude neem products in the control of culicine
[2] Gopalan SS, Das A. Household economic impact of an emerging
vectors of Japanese encephalitis and produced a slight but disease in terms of catastrophic out-of-pocket health care
significant reduction in population of anopheline pupae[42]. expenditure and loss of productivity: investigation of an outbreak
Dua et al stated that emulsified neem oil formulation showed of chikungunya in Orissa, India. J Vector Borne Dis 2009; 46(1):
95.5% reduction in larval population of Cx. quinquefasciatus 57-64.
on the first day under field trials and thereafter 80 % [3] Dhiman RC, Pahwa S, Dhillon GP, Dash AP. Climate change
reduction were achieved up to the third week[43]. In a recent and threat of vector-borne diseases in India: are we prepared?
study, the field trials were conducted by using Clerodendrum Parasitol Res 2010; 106(4): 763-773.
inerme and Acanthus ilicifolius in different habitats to [4] Senthilkumar N, Verma P, Gurusubamanian G. Larvicidal and
treat three species of mosquito vectors, namely, malarial adulticidal activities of some medicinal plants against the malarial
vector Anopheles stephensi (Liston). Parasitol Res 2009; 104(2):
vector An. stephensi, dengue vector Ae. aegypti, and filarial
237-244.
vector Cx. quinquefasciatus, in Vadavalli, Mettupalayam,
[5] Amer A, Mehlhorn H. Larvicidal effects of varies essential oils
Navavoor privu, Pommanam palayam, Ooty, Mettupalayam
against Aedes, Anopheles, and Culex larvae (Diptera, Culicidae).
(Kallaru) in Tamil Nadu, India. The percentage reduction of Parasitol Res 2006; 99(4): 466-472.
larval mortality also showed variations among the different [6] G ovindarajan M, Sivakumar R. Adulticidal and repellent
breeding habitats of mosquito vectors at 24, 48, and 72 h. properties of indigenous plant extracts against Culex
This may be due to the impact of geographical distribution quinquefasciatus and Aedes aegypti (Diptera: Culicidae). Parasitol
of An. stephensi, Ae. aegypti, and Cx. quinquefasciatus at Res 2012; 110(5): 1607-1620.
the breeding sites[44]. In field trial, Leucas aspera extract [7] Govindarajan M, Jebanesan A, Reetha D, Amsath R, Pushpanathan
had the highest percentage of larval mortality against Cx. T, Samidurai K. Antibacterial activity of Acalypha indica. L. Eur
quinquefasciatus, Abutilon indicum, Hyptis suaveolens and Rev Med Pharmacol Sci 2008; 12(5): 299-302.
[8] Govindarajan M, Jebanesan A, Pushpanathan T. Larvicidal and
Jatropha curcas extracts (60.4%, 51.7%, 50.0% and 46.7% at
ovicidal activity of Cassia fistula Linn leaf extract against filarial
24 h; 81.9%, 77.6%, 73.5% and 71.7% at 48 h; 99.7%, 92.0%,
and malarial vector mosquitoes. Parasitol Res 2008; 102(2): 289-
90.4% and 89.9% at 72 h)[45].
292.
In conclusion, the larvicidal potentiality of the crude [9] Kovendan K, Murugan K, Vincent S. Evaluation of larvicidal
extracts of C. roseus and B. thuringiensis was studied in activity of Acalypha alnifolia Klein ex Willd. (Euphorbiaceae) leaf
the laboratory as well as field conditions. The C. roseus leaf extract against the malarial vector, Anopheles stephensi, dengue
extract and B. thuringiensis have been shown to be effective vector, Aedes aegypti and Bancroftian filariasis vector, Culex
mosquito control agents. These results show that these two quinquefasciatus (Diptera: Culicidae). Parasitol Res 2012; 110(2):
biological agents could reduce the malarial incidence. It also 571-581.
divulges the presence of active metabolites which are causes [10]Kovendan K, Murugan K, Prasanna Kumar K, Panneerselvam C,
of larval mortality. Therefore, the botanicals are one of the Mahesh Kumar P, Amerasan D, et al. Mosquitocidal properties
of Calotropis gigantea (Family: Asclepiadaceae) leaf extract and
best alternatives for chemical insecticides and are also eco-
bacterial insecticide, Bacillus thuringiensis against the mosquito
friendly bio-pesticides which create a healthy environment.
vectors. Parasitol Res 2012; 111(2): 531-544.
[11]Collu G, Unver N, Peltenburg-looman AG, Van der Heijden
R, Verpoorte R, Memelink J. Geraniol 10-hydroxylase1, a
Conflict of interest statement cytochrome p450 enzyme involved in terpenoid indole alkaloid
biosynthesis. FEBS Lett 2001; 508(2): 215-220.
We declare that we have no conflict of interest. [12]Vimala Y, Jain R. A new flavone in mature Catharanthus roseus
 Chellasamy Panneerselvam et al./Asian Pacific Journal of Tropical Medicine (2013)857-853
853
petals. Indian J Plant Physiol 2001; 6(2): 187-189. http://nobelprize.org/medicine/educational/malaria. [Accessed on
[13]Hirose F, Ashihara H. Metabolic regulation in plant cell culture- April 24, 2009]
fine control of purine nucleotide biosynthesis in intact cells of [32]Karunamoorthi K, Ramanujam S, Rathinasamy R. Evaluation of
Catharanthus roseus. J Plant Physiol 1984; 116(5): 417-423. leaf extracts of Vitex negundo L. (Family: Verbenaceae) against
[14]L ikitvivatanavong S, Chen J, Bravo A, Soberon M, Gill SS. larvae of Culex tritaeniorhynchus and repellent activity on adult
Cadherin, alkaline phosphatase, and aminopeptidase N as vector mosquitoes. Parasitol Res 2008; 103(3): 545-550.
receptors of Cry11Ba toxin from Bacillus thuringiensis subsp. in [33]Mullai K, Jebanesan A, Pushpanathan T. Effect of bioactive
Aedes aegypti. Appl Environ Microbiol 2011; 77(1): 24-31. fractions of Citrullus vulgaris Schrad. leaf extract against
[15]Gill EA, McIntyre TM, Prescott SM, Zimmerman GA. Mechanisms Anopheles stephensi and Aedes aegypti. Parasitol Res 2008; 102(5):
of vascular injury in the pathogenesis of infectious-disease. Curr 951-955.
Opin Infect Dis 1992; 5(3): 381-388. [34]Rahuman AA, Gopalakrishnan G, Venkatesan P, Geetha K.
[16]de Maagd RA, Bosch D, Stiekema W. Bacillus thuringiensis toxin- Isolation and identification of mosquito larvicidal compound from
mediated insect resistance in plants. Trends Plant Sci 1999; 4(1): Abutilon indicum (Linn.) Sweet. Parasitol Res 2008; 102(5): 981-
9-13. 988.
[17]P oncet S, Delecluse A, Klier A, Rapoport G. Evaluation of [35]K ovendan K, Murugan K, Vincent S, Barnard DR. Studies
synergistic interactions among the CryIVa, CryIVb and CryIVd on larvicidal and pupicidal activity of Leucas aspera Willd.
toxic components of Bacillus thuringiensis subsp. israelensis (Lamiaceae) and bacterial insecticide, Bacillus sphaericus against
crystals. J Inverte Pathol 1995; 66(2): 131-135. malarial vector, Anopheles stephensi Liston. (Diptera: Culicidae).
[18]Pérez C, Fernandez LE, Sun J, Folch JL, Gill SS, Soberón M, Parasitol Res 2012; 110(1): 195-203.
et al. Bacillus thuringiensis subsp israelensis Cyt1Aa synergizes [36]Nathan SS, Kalaivani K, Murugan K. Effects of neem limonoids on
Cry11Aa toxin by functioning as a membrane-bound receptor. the malaria vector Anopheles stephensi Liston (Diptera: Culicidae).
Proc Natl Acad Sci U S A 2005; 102(51): 18303-18308. Acta Trop 2005; 96(1): 47-55.
[19]Wirth MC, Park HW, Walton WE, Federici BA. Cyt1A of Bacillus [37]Nathan SS, Kalaivani K, Murugan K. Behavioural responses and
thuringiensis delays evolution of resistance to Cry11A in the changes in biology of rice leaf-folder following treatment with
mosquito Culex quinquefasciatus. Appl Environ Microbiol 2005; a combination of bacterial toxins and botanical insecticides.
71(1): 185-189. Chemosphere 2006; 64(10): 1650-1658.
[20]Porter AG, Davidson EW, Liu JW. Mosquitocidal toxins of bacilli [38]Das K, Mukherjee AK. Assessment of mosquito larvicidal potency
and their genetic manipulation for effective biological control of of cyclic lipopeptides produced by Bacillus subtilis strains. Acta
mosquitoes. Microbiol Rev 1993; 57(4): 838-861. Trop 2006; 97(2):168-173.
[21]Becker N, Petric D, Zgomba M, Boase C, Dahl C, Kaiser A. [39]Kuppusamy C, Ayyadurai N. Synergistic activity of Cyt1A from
Mosquitoes and their control. Heidelberg: Springer; 2010, p. 577. Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus
[22]Gray EJ, Lee KD, Souleimanov AM, Falco MRD, Zhou X, Charles B101 H5a5b against Bacillus sphaericus B101 H5a5b-resistant
TC, et al. A novel bacteriocin, thuricin 17, produced by plant strains of Anopheles stephensi Liston (Diptera: Culicidae). Parasitol
growth promoting rhizobacteria strain Bt NEB17: isolation and Res 2012; 110(1): 381-388.
classification. J Appl Microbiol 2006; 100(3): 545-554. [40]Singh G, Prakash S. Efficacy of Bacillus sphaericus against larvae
[23]Medeiros FP, Santos MA, Regis L, Rios EM, Rolim Neto PJ. of malaria and filarial vectors: an analysis of early resistance
Development of a Bacillus sphaericus tablet formulation and detection. Parasitol Res 2009; 104(4): 763-766.
its evaluation as a larvicide in the biological control of Culex [41]M ahesh Kumar P, Murugan K, Kovendan K, Subramaniam
quinquefasciatus. Mem Inst Oswaldo Cruz 2005; 100(4): 431-434. J, Amerasan D. Mosquito larvicidal and pupicidal efficacy of
[24]Armengol G, Hernandez J, Velez JG, Orduz S. Long-lasting Solanum xanthocarpum (Family: Solanaceae) leaf extract and
effects of a Bacillus thuringiensis serovar israelensis experimental bacterial insecticide, Bacillus thuringiensis, against Culex
tablet formulation for Aedes aegypti (Diptera: Culicidae) control. J quinquefasciatus Say. (Diptera: Culicidae). Parasitol Res 2012;
Econ Entomol 2006; 99(5): 1590-1595. 110(6): 2541-2550.
[25]Balaraman K, Gunasekaran K, Pillai PK, Manonmani AM. Field [42]Rao DR, Reuben R, Nagasampagi BA. Development of combined
trial with different formulations of Bacillus sphaericus for mosquito use of neem (Azadirachta indica) and water management for the
control. Indian J Med Res 1987; 85: 620-625. control of culicine mosquitoes in rice fields. Med Vet Entomol
[26]Geetha I, Manonmani AM. Surfactin: a novel mosquitocidal 1995; 9(1): 25-33.
biosurfactant produced by Bacillus subtilis sp. subtilis (VCRC [43]Dua VK, Pandey AC, Raghavendra K, Gupta A, Sharma T, Dash
B471) and influence of abiotic factors on its pupicidal efficacy. Let AP. Larvicidal activity of neem oil (Azadirachta indica formulation
Appl Microbial 2010; 51(4): 406-412. against mosquitoes). Malar J 2009; 8: 124.
[27]Vogel AI. Textbook of practical organic chemistry. London: The [44]K ovendan K, Murugan K. Effect of medicinal plants on the
English Language Book Society and Longman; 1978, p. 1368. mosquito vectors from the different agro-climatic regions of Tamil
[28]A bbott WS. A method of computing the effectiveness of Nadu, India. Adv Environ Biol 2011; 5(2): 335-344.
insecticides. 1925. J Am Mosq Control Assoc 1987; 3(2): 302-303. [45]Kovendan K, Murugan K, Panneerselvam C, Mahesh Kumar
[29]Finney DJ. Probit analysis. London: Cambridge University; 1971, P, Amerasan D, Subramaniam J, et al. Laboratory and field
p. 68-78. evaluation of medicinal plant extracts against filarial vector, Culex
[30]Alder HL, Rossler EB. Introduction to probability and statistics. quinquefasciatus Say (Diptera: Culicidae). Parasitol Res 2012;
San Francisco: Freeman; 1977, p. 246. 110(6): 2105-2115.
[31]Lambert PH. Malaria: past and present. [Online]. Available from: