Parasitol Res (2011) 109:353–367

DOI 10.1007/s00436-011-2263-1

ORIGINAL PAPER

Mosquito larvicidal, ovicidal, and repellent properties

of botanical extracts against Anopheles stephensi, Aedes aegypti,
and Culex quinquefasciatus (Diptera: Culicidae)
M. Govindarajan & T. Mathivanan & K. Elumalai &
K. Krishnappa & A. Anandan

Received: 25 December 2010 / Accepted: 18 January 2011 / Published online: 12 February 2011
# Springer-Verlag 2011

Abstract Mosquito-borne diseases have an economic vector mosquitoes. For E. coronaria, the benzene extract
impact, including loss in commercial and labor outputs, exerted 300, 250, and 200 ppm against Anopheles
particularly in countries with tropical and subtropical stephensi, Aedes aegypti, and Culex quinquefasciatus,
climates; however, no part of the world is free from respectively. The results of the repellent activity of benzene
vector-borne diseases. In mosquito control programs, and ethyl acetate extract of E. coronaria and Caesalpinia
botanical origin may have the potential to be used pulcherrima plants at three different concentrations of 1.0,
successfully as eggs, larvae, and adult. The larvicidal, 2.5, and 5.0 mg/cm2 were applied on skin of fore arm in
ovicidal, and repellent activities of crude benzene and ethyl man and exposed against adult female mosquitoes. In this
acetate extracts of leaf of Ervatamia coronaria and observation, these two plant crude extracts gave protection
Caesalpinia pulcherrima were assayed for their toxicity against mosquito bites without any allergic reaction to the
against three important vector mosquitoes, viz., Anopheles test person, and also, the repellent activity is dependent on
stephensi, Aedes aegypti, and Culex quinquefasciatus the strength of the plant extracts. These results suggest that
(Diptera: Culicidae). The larval mortality was observed the leaf solvent plant extracts have the potential to be used
after 24 h of exposure. All extracts showed moderate as an ideal ecofriendly approach for the control of
larvicidal effects; however, the highest larval mortality was mosquitoes. This is the first report on the mosquito
found in benzene extract of E. coronaria against the larvae larvicidal, ovicidal, and repellent activities of the reported
of Anopheles Stephensi, Aedes aegypti, and Culex quinque- E. coronaria and Caesalpinia pulcherrima plants.
fasciatus with the LC50 and LC90 values were 79.08, 89.59,
and 96.15 ppm and 150.47, 166.04, and 174.10 ppm,
respectively. Mean percent hatchability of the ovicidal Introduction
activity was observed 48 h posttreatment. The percent
hatchability was inversely proportional to the concentration Mosquitoes are the major vector for the transmission of
of extract and directly proportional to the eggs. The leaf malaria, dengue fever, yellow fever, filariasis, schistosomi-
extract of E. coronaria was found to be most effective than asis, Japanese encephalitis (JE), etc., causing millions of
Caesalpinia pulcherrima against eggs/egg rafts of three deaths every year (James 1992). Mosquitoes also cause
allergic responses in humans that include local skin and
M. Govindarajan (*) : T. Mathivanan : K. Krishnappa : systemic reactions such as angioedema (Peng et al. 1999).
A. Anandan Aedes aegypti (L.) is generally known as a vector for an
Division of Vector Biology and Phytochemistry,
arbovirus responsible for dengue fever, which is endemic to
Department of Zoology, Annamalai University,
Annamalai Nagar 608 002, Tamil Nadu, India Southeast Asia, the Pacific island area, Africa, and the
e-mail: drgovind1979@gmail.com Americas. This mosquito is also the vector of yellow fever
in Central and South America and West Africa. Dengue
K. Elumalai
fever has become an important public health problem as the
Unit of Entomotoxicity, Department of Zoology,
Government Thirumagal Mills College, number of reported cases continues to increase, especially
Vellore 632 607, Tamil Nadu, India with more severe forms of the disease, dengue hemorrhagic
354 Parasitol Res (2011) 109:353–367

fever, and dengue shock syndrome, or with unusual the effectiveness of plant extracts or essential oils against
manifestations such as central nervous system involvement mosquito larvae (Amer and Mehlhorn 2006a, b, c;
(Pancharoen et al. 2002). Culex quinquefasciatus (Say.) acts Govindarajan 2010a, b).
as a vector for filariasis in India. Human filariasis is a major Phytochemicals are advantageous due to their eco-safety,
public health hazard and remains a challenging socioeco- target-specificity, nondevelopment of resistance, reduced
nomic problem in many of the tropical countries (Udonsi number of applications, higher acceptability, and suitability
1986). Lymphatic filariasis caused by Wuchereria bancrofti for rural areas. Botanicals can be used as alternative to
and transmitted by mosquito Culex quinquefasciatus is synthetic insecticides or along with other insecticides under
found to be more endemic in the Indian subcontinent. It is integrated vector control programs. The plant product of
reported that Culex quinquefasciatus infects more than 100 phytochemical, which is used as insecticides for killing
million individuals worldwide annually (Rajasekariah et al. larvae or adult mosquitoes or as repellents for protection
1991). W. bancrofti is the most predominant filarial against mosquito bites. Phytochemicals obtained from the
nematode, which is usually characterized by progressive whole plant or specific part of the plant by the extraction
debilitating swelling at the extremities, scrotum, or breast with different types of solvent such as aqueous, methanol,
(elephantiasis) in an infected individual (Myung et al. chloroform, benzene, acetone, etc., depending on the
1998). Anopheles stephensi Liston is the primary vector of polarity of the phytochemical. Some phytochemicals act
malaria in India and other West Asian countries, Malaria as toxicant (insecticide) both against adult as well as larval
remains one of the most prevalent diseases in the tropical stages of mosquitoes, while others interfere with growth
world. With 200 million to 450 million infections annually and growth inhibitor or with reproduction or produce an
worldwide, it causes up to 2.7 million deaths (World Health olfactory stimulus, thus acting as repellent or attractant
Organization 2010). The disease remains endemic in more (Markouk et al. 2001).
than 100 developing tropical countries, and its control is a Plants may be a source of alternative agents for control
major goal for improved worldwide health. of mosquitoes because they are rich in bioactive chemicals,
Mosquito control has been becoming increasingly are active against a limited number of species including
difficult because of the indiscriminate uses of synthetic specific target insects, and are biodegradable. They are
chemical insecticides which have an adverse impact on the potentially suitable for use in integrated pest management
environment and disturb ecological balance. Majority of the programs (Alkofahi et al. 1989): the mosquito larvicidal
chemical pesticides are harmful to man and animals, some properties of leaf and seed extract of plant Agave
of which are not easily degradable and spreading toxic Americana (Dharmshaktu et al. 1987); the mosquito
effects. The increased use of these insecticides may enter larvicidal activity in the extract of Tagetes minuta flowers
into the food chain, and thereby, the liver, kidney, etc., may against Aedes aegypti (Green et al. 1991); the methanolic
be irreversibly damaged. They even result in mutation of fraction of leaves of Mentha piperita, Phyllanthus niruri,
genes and these changes become prominent only after a few Leucas aspera, and Vitex negundo against larvae of Culex
generations (Ghosh 1991). Chemical insecticides is very quinquefasciatus (Pandian et al. 1994); the methanolic
costly. In larval mosquito control, application of insecti- extracts of Solanum suratense, Azadirachta indica, and
cides in ponds, wells, and other water bodies may cause Hydrocotyle javanica exhibited larvicidal activity against
health hazards to human and larvivorus fishes. Nowadays, Culex quinquefasciatus (Muthukrishnan et al. 1997); the
mosquito coils containing synthetic pyrethroids and other benzene and methanol extracts of Artemisia vulgaris has
organophosphorus compounds cause so many side effects, been repellent activity against Aedes Aegypti (Yit et al.
such as breathing problem, eye irritation, headache, asthma, 1985); the Zanthoxylum armatus, Zanthoxylum alatum
itching, and sneezing to the users. With the use mosquito (Rutaceae), Azadirachta indica (Mailiaceae), and Curcuma
repellent, people complained of ill health effect and aromatica (Zingiberaceae) were possessing repellent prop-
sometimes required medical treatment. In addition, pests erties against mosquitoes (Das et al. 2000); the repellent
were becoming resistant to chemical treatments. Indoor activity of active compound Octacosane from Moschosma
residual spraying of insecticides stains the walls and leaves polystachyum against the vector Culex quinquefasciatus
a long lasting unpleasant odor. These problems have (Rajkumar and Jebanesan 2004); and the essential oil of
highlighted the need for the development of new strategies Zingiber officinalis as a mosquito larvicidal and repellent
for selective mosquito control. Extracts or essential oils agent against the filarial vector Culex quinquefasciatus
from plants may be alternative sources of mosquito larval (Pushpanathan et al. 2008).
control agents, since they constitute a rich source of Today, more than 1,005 plant species are found to
bioactive compounds that are biodegradable into nontoxic possess insecticidal properties, 384 contain antifeedants,
products and potentially suitable for use in control of 297 contain repellents, and 27 contain attractants and
mosquito larvae. In fact, many researchers have reported on possess growth inhibitors (Jayaraj 1993). Larvicidal effica-
Parasitol Res (2011) 109:353–367 355

cy of the crude leaf extract of Ficus benghalensis with three voucher specimen is deposited at the herbarium of plant
different solvents like methanol, benzene, and acetone were phytochemistry division, Department of Zoology, Annamalai
tested against the early second, third, and fourth instar University.
larvae of Culex quinquefasciatus, Aedes aegypti, and
Anopheles stephensi (Govindarajan 2010c). The acetone, Extraction
chloroform, ethyl acetate, hexane, and methanol leaf
extracts of Acalypha indica, Achyranthes aspera, L. The leaves were washed with tap water, shade-dried, and
aspera, Morinda tinctoria, and Ocimum sanctum were finely ground. The finely ground plant leaf powder (3.0 kg/
studied against the early fourth instar larvae of Aedes solvent) was loaded in Soxhlet apparatus and was extracted
aegypti and Culex quinquefasciatus (Bagavan et al. with two different solvents, namely, benzene and ethyl
2008a). Larvicidal activity of crude hexane, ethyl acetate, acetate, individually (Vogel 1978). The solvents from the
petroleum ether, acetone, and methanol extracts of the leaf extracts were removed using a rotary vacuum evaporator to
of five species of cucurbitaceous plants, Citrullus colo- collect the crude extract. Standard stock solutions were
cynthis, Coccinia indica, Cucumis sativus, Momordica prepared at 1% by dissolving the residues in acetone. From
charantia, and Trichosanthes anguina, were tested against this stock solution, different concentrations were prepared
the early fourth instar larvae of Aedes aegypti L. and Culex and these solutions were used for larvicidal, ovicidal, and
quinquefasciatus (Rahuman et al. 2008). Govindarajan repellent bioassays.
(2009) reported that the leaf methanol, benzene, and
acetone extracts of Cassia fistula were studied for the Test organisms
larvicidal, ovicidal, and repellent activities against Aedes
aegypti. The leaf extract of Acalypha indica with different The mosquitoes, Anopheles stephensi, Aedes aegypti, and
solvents, viz., benzene, chloroform, ethyl acetate, and Culex quinquefasciatus were reared in the vector control
methanol, was tested for larvicidal, ovicidal activity, and laboratory, Department of Zoology, Annamalai University.
oviposition attractancy against Anopheles stephensi The larvae were fed on dog biscuits and yeast powder in the
(Govindarajan et al. 2008a). Mullai et al. (2008) have 3:1 ratio. Adults were provided with 10% sucrose solution
reported that the leaf extract of Citrullus vulgaris with and 1-week-old chick for blood meal. Mosquitoes were
different solvents, viz., benzene, petroleum ether, ethyl held at 28±2°C, 70–85% relative humidity, with a photo
acetate, and methanol, were tested for larvicidal, ovicidal, period of 14-h light and 10-h dark.
repellent, and insect growth regulatory activities against
Anopheles stephensi. Ovicidal effects of the seed extract of Larvicidal bioassay
Atriplex canescens was reported against Culex quinque-
fasciatus (Ouda et al. 1998). Su and Mulla (1998) reported The larvicidal activity of the plants crude extracts was
the ovicidal activity of the Neem product azadirachtin evaluated as per the method recommended by World Health
against the mosquitoes Culex tarsalis and Culex quinque- Organization (2005). Batches of 25 third instar larvae were
fasciatus. In view of the recently increased interest in transferred to a small disposable test cups, each containing
developing plant origin insecticides as an alternative to 200 ml of water. The appropriate volume of dilution was
chemical insecticide, this study was undertaken to assess added to 200 ml water in the cups to obtain the desired
the larvicidal, ovicidal, and repellent potential of the target dosage, starting with the lowest concentration. Six
different solvent crude extracts from the medicinal plants replicates were set up for each concentration, and an equal
Ervatamia coronaria and Caesalpinia pulcherrima against number of controls were set up simultaneously using tap
the medically important mosquito vectors, Culex quinque- water. To this, 1 ml of appropriate solvent was added. The
fasciatus, Aedes aegypti, and Anopheles Stephensi. LC50 value was calculated after 24 h by probit analysis
(Finney 1979).

Materials and methods Ovicidal activity

Collection of plants For ovicidal activity, slightly modified method of Su and

Mulla (1998) was performed. The eggs/egg rafts of
Fully developed leaves of the Ervatamia coronaria and Anopheles stephensi, Aedes aegypti, and Culex quinque-
Caesalpinia pulcherrima were collected from in and around fasciatus were collected from vector control laboratory,
Annamalai University Campus, Annamalainagar, Tamil Annamalai University. The different leaf extracts diluted in
Nadu, India. It was authenticated by a plant taxonomist the appropriate solvent to achieve various concentrations
from the Department of Botany, Annamalai University. A ranging from 50 to 450 ppm. Eggs of these mosquito
356 Parasitol Res (2011) 109:353–367

species (100) were exposed to each concentration of leaf shaken off before imbibing any blood, making out a 5-min
extracts. After treatment, the eggs from each concentration protection. The percentage of repellency was calculated by
were individually transferred to distilled water cups for the following formula:% Repellency ¼½ðTa Tb Þ=Ta Š
hatching assessment after counting the eggs under micro- 100; where Ta is the number of mosquitoes in the control
scope. Each experiment was replicated six times along with group and Tb is the number of mosquitoes in the treated
appropriate control. The hatch rates were assessed 48 h group.
posttreatment by following formula.
Statistical analysis
Number of hatched larvae
% of egg mortality ¼  100
Total no: of eggs The average larval mortality data were subjected to probit
analysis for calculating LC50, LC90, and other statistics at
95% confidence limits of upper confidence limit (UCL) and
Repellent activity lower confidence limit (LCL), and chi-square values were
calculated using the SPSS12.0 (Statistical Package of
The repellent study was following the method of World Social Sciences) software. Results with p<0.05 were
Health Organization (2009). Three-day-old blood-starved considered to be statistically significant.
female Culex quinquefasciatus, Aedes aegypti, and Anoph-
eles stephensi mosquitoes (100) were kept in a net cage
(45×30×45 cm). The volunteer had no contact with lotions, Results
perfumes, or perfumed soaps on the day of the assay. The
arms of volunteer, only 25 cm2 dorsal side of the skin on Table 1 shows the list of medicinal plants tested for the
each arms was exposed and the remaining area covered by bioactivity against vector mosquitoes. The results of the
rubber gloves. The crude extract was applied at 1.0, 2.5, larvicidal activity of benzene and ethyl acetate extract of
and 5.0 mg/cm2 separately in the exposed area of the fore Ervatamia coronaria and Caesalpinia pulcherrima against
arm. Only ethanol served as control. The time of the test is the larvae of three important vector mosquitoes, viz.,
dependent on whether the target mosquitoes are day or Anopheles stephensi, Aedes aegypti, and Culex quinque-
night biters. Aedes aegypti was tested during the day time fasciatus, are presented in Tables 2 and 3 and Fig. 1.
from 07:00 to 17:00 hours, while Culex quinquefasciatus Among two plants tested, the highest larvicidal activity was
and Anopheles stephensi were tested during the night from observed in benzene extract of Ervatamia coronaria against
19:00 to 05:00 hours. The control and treated arms were Anopheles stephensi than Aedes aegypti and Culex quin-
introduced simultaneously into the mosquito cage, and quefasciatus, with the LC50 and LC90 values were 79.08,
gently tapping the sides on the experimental cages, the 89.59, and 96.15 ppm and 150.47, 166.04, and 174.10 ppm,
mosquitoes were activated. Each test concentration was respectively. The chi-square values are significant at p<
repeated six times. The volunteer conducted their test of 0.05 level. The chi-square values in the bioassays indicated
each concentration by inserting the treated and control arms probably the heterogeneity of the test population. The 95%
in to the same cage for one full minute for every 5 min. The confidence limits (LC50 (LCL–UCL)) and (LC90 (LCL–
mosquitoes that landed on the hand were recorded and then UCL)) were also calculated. The percentage of egg

Table 1 List of medicinal plants tested for the bioactivity against vector mosquitoes

Botanical name Common name (Tamil) Family Medicinal property Plant parts tested

Ervatamia coronaria Nanthiyavattai Apocynaceae To make perfume and, medicinally, to Leaves

(Jacq.) Stapf. provide a refrigerant (cooling) drink.
A decoction of the roots is used in India
to stop diarrhea. Pulp surrounding the
seed is used in the Himalayan area to
produce a red dye.
Caesalpinia pulcherrima Mayurkonrai Fabaceae Plant pacifies vitiated kapha, pitta, fever, Leaves
(Linn.) Swartz. jaundice, colic, flatulence, malignant
tumors. It is a proven anti-cancerous drug.
In overdose, it shows toxic symptoms and
the seeds are said to cure bad cough,
breathing difficulty, and chest pain. Four
grams from the root is also said to induce
abortion in the first trimester of pregnancy.
Parasitol Res (2011) 109:353–367 357

Table 2 Larvicidal activity of crude benzene and ethyl acetate extracts of Ervatamia coronaria against Culex quinquefasciatus, Aedes aegypti,
and Anopheles stephensi

Solvents Mosquitoes Concentration % of mortality ± SD LC50 (LCL–UCL) LC90 (LCL–UCL) χ2

(mg/L) (mg/L) (mg/L)

Benzene Culex quinquefasciatus 30 18.9±1.2 96.15 (89.50–102.86) 174.10 (162.99–187.95) 10.618a

60 31.3±1.8
90 48.9±2.2
120 63.6±1.4
150 77.0±1.6
180 92.6±1.2
Control 0.0±0.0
Aedes aegypti 30 22.3±1.6 89.59 (74.42–104.60) 166.04 (144.84–200.63) 14.295a
60 35.0±1.3
90 54.0±1.4
120 66.3±1.7
150 79.6±1.9
180 95.3±2.2
Control 0.0±0.0
Anopheles stephensi 30 27.0±0.8 79.08 (60.06–96.76) 150.47 (127.57–191.80) 21.897a
60 41.3±1.2
90 62.6±1.9
120 71.0±1.5
150 84.6±1.4
180 99.3±1.9
Control 0.0±0.0
Ethyl acetate Culex quinquefasciatus 30 18.0±1.2 110.37 (92.53–130.09) 176.53 (157.84–236.10) 24.440a
60 29.0±0.8
90 38.6±1.9
120 51.0±1.7
150 71.0±1.3
180 95.0±1.4
Control 0.0±0.0
Aedes aegypti 30 25.3±1.6 97.53 (75.83–120.73) 179.37 (149.17–241.99) 26.009a
60 32.0±1.4
90 41.6±1.3
120 57.9±1.9
150 73.3±1.7
180 96.6±2.2
Control 0.0±0.0
Anopheles stephensi 30 26.3±2.4 88.92 (67.87–109.70) 168.05 (140.68–221.82) 24.376a
60 36.0±1.8
90 54.6±1.7
120 61.3±1.3
150 78.0±1.6
180 97.6±1.9
Control 0.0±0.0

Each value (X  S:D:) represents mean of six values

LCL lower confidence limit, UCL upper confidence limit
a
Significant at p<0.05 level
358 Parasitol Res (2011) 109:353–367

Table 3 Larvicidal activity of crude benzene and ethyl acetate extracts of Caesalpinia pulcherrima against Culex quinquefasciatus, Aedes
aegypti, and Anopheles stephensi

Solvents Mosquitoes Concentration % of mortality±SD LC50 (LCL–UCL) LC90 (LCL–UCL) χ2

(mg/L) (mg/L) (mg/L)

Benzene Culex quinquefasciatus 50 26.0±1.8 150.65 (119.09–182.06) 287.55 (244.08–367.35) 19.394a

100 38.3±1.4
150 47.6±1.9
200 66.6±1.3
250 76.6±2.2
300 94.6±1.5
Control 0.0±0.0
Aedes aegypti 50 32.0±1.7 136.36 (98.79–171.22) 272.15 (226.16–363.29) 24.702a
100 44.0±1.5
150 51.3±1.6
200 72.0±1.3
250 80.6±2.2
300 96.0±1.8
Control 0.0±0.0
Anopheles stephensi 50 34.3±1.2 126.56 (85.41–162.76) 258.23 (212.18–352.67) 28.194a
100 48.6±1.8
150 54.0±1.4
200 78.0±2.2
250 82.6±1.9
300 97.0±1.2
Control 0.0±0.0
Ethyl acetate Culex quinquefasciatus 50 23.3±0.8 154.33 (127.40–181.48) 288.08 (249.35–353.62) 15.247a
100 35.6±1.2
150 47.6±1.4
200 65.3±1.6
250 77.3±1.4
300 93.9±1.2
Control 0.0±0.0
Aedes aegypti 50 27.3±1.2 144.67 (114.75–173.77) 276.99 (236.81–347.85) 18.097a
100 38.0±1.8
150 51.6±1.3
200 68.6±1.5
250 80.0±1.8
300 95.3±1.2
Control 0.0±0.0
Anopheles stephensi 50 30.6±1.8 130.90 (99.57–159.96) 253.85 (215.56–322.10) 20.480a
100 42.0±1.4
150 58.0±1.6
200 72.3±2.2
250 86.0±0.8
300 98.0±1.9
Control 0.0±0.0

Each value (X  S:D:) represents mean of six values

LCL lower confidence limit, UCL upper confidence limit
a
Significant at P<0.05 level
Parasitol Res (2011) 109:353–367 359

Fig. 1 Graph showing the LC50

and LC90 values of three
important vector mosquitoes

Table 4 Ovicidal activity of Ervatamia coronaria plant extracts against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi

Mosquito Name of the solvent Percentage of egg hatch ability

Concentration (mg/L)

Control 50 100 150 200 250 300

Culex quinquefasciatus Benzene 100±0.0 75.4±0.8 54.8±1.2 38.6±1.5 32.4±1.2 21.6±1.5 NH

Ethyl acetate 100±0.0 86.2±1.2 69.6±1.5 51.2±1.2 38.6±1.7 25.4±1.8 NH
Aedes aegypti Benzene 100±0.0 68.3±1.7 49.6±1.9 35.8±1.8 28.4±1.8 NH NH
Ethyl acetate 100±0.0 81.6±1.3 67.4±2.2 46.6±2.2 35.2±1.2 22.8±1.6 NH
Anopheles stephensi Benzene 100±0.0 61.8±1.4 46.2±1.4 29.0±1.4 NH NH NH
Ethyl acetate 100±0.0 77.6±1.2 62.4±1.6 42.2±1.6 31.4±1.6 NH NH

Each value (X  S:D:) represents the mean of six values.

NH no hatchability (100% mortality)
360 Parasitol Res (2011) 109:353–367

Table 5 Ovicidal activity of Caesalpinia pulcherrima plant extracts against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi

Mosquito Name of the solvent Percentage of egg hatch ability

Concentration (mg/L)

Control 75 150 225 300 375 450

Culex quinquefasciatus Benzene 100±.0.0 82.2±2.2 67.6±1.9 53.8±2.2 38.6±1.5 24.6±1.2 NH

Ethyl acetate 100±.0.0 95.4±1.8 78.6±1.7 57.6±1.5 41.4±1.3 29.4±1.8 NH
Aedes aegypti Benzene 100±.0.0 71.4±1.7 56.8±0.8 42.4±1.8 26.8±1.8 NH NH
Ethyl acetate 100±.0.0 86.4±1.3 68.4±1.2 51.6±1.9 34.8±1.2 22.4±1.6 NH
Anopheles stephensi Benzene 100±.0.0 88.3±1.8 52.7±1.4 39.2±1.3 NH NH NH
Ethyl acetate 100±.0.0 79.4±1.6 61.8±1.8 48.6±1.9 31.4±0.8 NH NH

Each value (X  S:D:) represents the mean of six values

NH no hatchability (100% mortality)

hatchability of Culex quinquefasciatus, Aedes aegypti, and expensive imported products, and stimulate local efforts to
Anopheles stephensi were tested with two different solvents enhance public health. Different parts of plants contain a
at different concentrations of Ervatamia coronaria and complex of chemicals with unique biological activity
Caesalpinia pulcherrima plant leave extracts, and the (Govindarajan et al. 2008a, b, c), which is thought to be
results are listed in Tables 4 and 5. The percent hatchability due to toxins and secondary metabolites which act as
was inversely proportional to the concentration of extract mosquitocidal agent (Niraimathi et al. 2010). Our result
and directly proportional to the eggs. Among the two plants showed that the crude benzene and ethyl acetate extracts of
leaves extracts tested for ovicidal activity against Anopheles leaf of Ervatamia coronaria and Caesalpinia pulcherrima
stephensi, Aedes aegypti, and Culex quinquefasciatus, the have significant larvicidal, ovicidal, and repellent properties
benzene extract of Ervatamia coronaria exerted 100% against three important vector mosquitoes, viz., Anopheles
mortality (zero hatchability) at 300, 250, and 200 ppm, stephensi, Aedes aegypti, and Culex quinquefasciatus. This
respectively. The leaf extract of Ervatamia coronaria was result is also comparable to earlier reports of Singh et al.
found to be most effective than Caesalpinia pulcherrima (2003) who observed the larvicidal activity of Ocimum
against eggs of three vector mosquitoes. Control eggs canum oil against vector mosquitoes, namely, Aedes
showed the 100% hatchability. The benzene and ethyl aegypti and Culex quinquefasciatus (LC50, 301 ppm) and
acetate extracts of leaves of Ervatamia coronaria and Anopheles stephensi (234 ppm). Larvicidal efficacy of leaf
Caesalpinia pulcherrima show significant repellency methanol extracts of Pavonia zeylanica and Acacia ferru-
against Anopheles stephensi, Aedes aegypti, and Culex ginea were tested against the late third instar larvae of
quinquefasciatus (Tables 6 and 7) In this observation, these Culex quinquefasciatus with LC50 values of 2,214.7 and
two plant crude extracts gave protection against mosquito 5,362.6 ppm, respectively (Vahitha et al. 2002). The peel
bites without any allergic reaction to the test person, and methanol extract of Citrus sinensis and the leaf and flower
also, the repellent activity is dependent on the strength of ethyl acetate extracts of O. canum were tested against the
the plant extracts. larvae of Anopheles stephensi (LC50 =95.74, 101.53, and
28.96 ppm; LC90 = 303.20, 492.43 and 168.05 ppm),
respectively (Kamaraj et al. 2008a). Karunamoorthi et al.
Discussions (2008) reported that the petroleum ether (60–80°C) extracts
of the leaves of V. negundo were evaluated for larvicidal
Today, the environmental safety of an insecticide is activity against larval stages of Culex tritaeniorhynchus in
considered to be of paramount importance. An insecticide the laboratory with LC50 and LC90 values of 2.4883 and
does not have to cause high mortality on target organisms 5.1883 mg/l, respectively. The methanol leaf extracts of V.
in order to be acceptable (Kabaru and Gichia 2001). negundo, Vitex trifolia, Vitex peduncularis, and Vitex
Phytochemicals may serve as suitable alternatives to altissima possessed varying levels of larvicidal activity on
synthetic insecticides in future as they are relatively safe Culex quinquefasciatus and Anopheles stephensi and were
and inexpensive and are readily available in many areas of found with LC50 value of 212.57, 41.41, 76.28, and
the world. According to Bowers et al. (1995), the screening 128.04 ppm, respectively (Pushpalatha and Muthukrishnan
of locally available medicinal plants for mosquito control 1995). The ethanolic leaf extract of Cassia obtusifolia had
would generate local employment, reduce dependence on significant larvicidal effect against Anopheles stephensi with
 Parasitol Res (2011) 109:353–367
Table 6 Repellent activity of crude benzene and ethyl acetate extract of Ervatamia coronaria against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi

Solvents Mosquitoes Concentration (mg/cm2) % of repellency

Time post application of repellent (min)

30 60 90 120 150 180 210 240

Benzene Culex quinquefasciatus 1.0 100±0.0 100±0.0 89.3±2.2 72.5±1.2 69.7±1.8 58.5±1.5 44.3±1.9 36.0±1.5
2.5 100±0.0 100±0.0 100±0.0 85.3±1.6 78.2±1.6 71.7±1.2 59.4±1.3 45.5±1.2
5.0 100±0.0 100±0.0 100±0.0 100±0.0 86.4±1.3 80.4±1.3 74.9±1.4 66.3±1.8
Aedes aegypti 1.0 100±0.0 100±0.0 100±0.0 86.9±0.8 74.5±1.7 64.0±1.8 56.7±1.6 48.0±1.4
2.5 100±0.0 100±0.0 100±0.0 100±0.0 82.0±1.5 79.1±1.3 68.6±1.7 59.2±1.6
5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 86.9±1.7 81.0±1.9 72.8±1.2
Anopheles stephensi 1.0 100±0.0 100±0.0 100±0.0 100±0.0 84.0±1.2 72.5±1.2 62.4±1.2 54.4±1.5
2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 88.4±1.6 74.8±1.4 62.8±1.2
5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 88.3±1.9 79.0±1.8
Ethyl acetate Culex quinquefasciatus 1.0 100±0.0 87.3±0.8 74.0±1.2 68.7±1.5 59.6±0.8 51.0±1.4 49.0±1.9 31.2±1.3
2.5 100±0.0 100±0.0 85.4±1.5 79.9±1.8 71.4±1.4 64.4±1.6 58.3±1.6 38.8±1.6
5.0 100±0.0 100±0.0 100±0.0 88.2±1.3 80.2±1.6 71.6±1.9 44.5±1.3 46.4±1.7
Aedes aegypti 1.0 100±0.0 100±0.0 75.0±1.9 61.4±1.7 61.8±1.8 53.2±1.2 44.3±1.5 38.3±1.5
2.5 100±0.0 100±0.0 100±0.0 74.2±1.5 74.9±1.6 66.8±1.8 54.6±2.1 47.2±1.8
5.0 100±0.0 100±0.0 100±0.0 100±0.0 81.0±1.2 79.1±1.5 68.2±1.8 59.8±1.5
Anopheles stephensi 1.0 100±0.0 100±0.0 100±0.0 74.0±1.6 69.1±0.8 61.2±1.7 57.2±0.9 52.7±1.2
2.5 100±0.0 100±0.0 100±0.0 100±0.0 81.5±1.9 76.4±1.3 69.7±1.7 61.1±1.9
5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 84.9±2.1 77.9±1.2 73.6±2.2

Each value (X  S:D:) represents mean of six values

361
 362
Table 7 Repellent activity of crude benzene and ethyl acetate extract of Caesalpinia pulcherrima against Culex quinquefasciatus, Aedes aegypti, and Anopheles stephensi

Solvents Mosquitoes Concentration (mg/cm2) % of repellency

Time post application of repellent (min)

15 30 60 90 120 150 180 210

Benzene Culex quinquefasciatus 1.0 100±0.0 100±0.0 77.0±0.8 69.9±1.2 57.6±1.4 44.0±1.8 32.2±1.2 26.5±1.5
2.55.0 100±0.0 100±0.0 100±0.0 79.2±1.4 65.4±1.9 53.8±1.4 49.1±1.6 31.9±1.7
100±0.0 100±0.0 100±0.0 100±0.0 79.2±1.8 68.4±2.2 56.8±1.9 43.1±1.3
Aedes aegypti 1.0 100±0.0 100±0.0 100±0.0 74.5±0.8 66.1±1.8 54.9±0.9 42.4±1.8 37.8±0.9
2.5 100±0.0 100±0.0 100±0.0 100±0.0 76.8±1.5 65.5±1.3 53.7±1.5 46.1±1.8
5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 77.6±1.8 69.9±1.7 58.3±1.9
Anopheles stephensi 1.0 100±0.0 100±0.0 100±0.0 100±0.0 79.2±1.4 68.0±1.3 56.2±1.4 48.8±1.5
2.5 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 81.5±1.6 71.3±1.8 62.1±1.7
5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 82.0±1.6 71.6±2.2
Ethyl acetate Culex quinquefasciatus 1.0 100±0.0 79.0±0.8 71.5±1.2 58.5±1.4 47.1±1.8 35.0±1.2 29.2±0.8 21.0±1.6
2.5 100±0.0 100±0.0 78.4±1.5 69.9±1.6 52.6±1.9 49.5±1.9 33.5±1.5 26.6±1.8
5.0 100±0.0 100±0.0 100±0.0 74.7±2.2 68.4±1.8 57.7±2.2 46.8±1.7 41.4±1.4
Aedes aegypti 1.0 100±0.0 100±0.0 68.9±0.9 61.1±1.9 58.9±1.4 46.4±1.4 35.0±1.2 29.8±1.5
2.5 100±0.0 100±0.0 100±0.0 72.6±1.5 69.8±1.6 54.6±1.8 48.9±1.6 38.7±1.8
5.0 100±0.0 100±0.0 100±0.0 100±0.0 73.6±1.9 66.3±1.9 57.4±2.1 49.1±1.4
Anopheles stephensi 1.0 100±0.0 100±0.0 100±0.0 71.0±1.6 67.3±1.2 54.1±1.4 46.6±1.6 38.0±1.2
2.5 100±0.0 100±0.0 100±0.0 100±0.0 76.0±1.5 68.5±1.5 57.7±1.7 49.2±1.8
5.0 100±0.0 100±0.0 100±0.0 100±0.0 100±0.0 71.9±1.8 69.3±1.9 58.9±2.2

Each value (X  S:D:) represents mean of six values

Parasitol Res (2011) 109:353–367

Parasitol Res (2011) 109:353–367 363

LC50 and LC90 values of 52.2 and 108.7 mg/l, respectively Aedes aegypti (LC50, 82.1 ppm), Culex quinquefasciatus
(Rajkumar and Jebanesan 2009); the emodin compound was (LC 50, 85.7 ppm), and Anopheles stephensi (LC 50 ,
isolated from seeds and showed the LC50 values of 1.4, 1.9, 112.6 ppm). Cavalcanti et al. (2004) reported that the
and 2.2 mg/L against Culex pipiens, Aedes aegypti, and larvicidal activity of essential oils of Brazilian plants
Aedes togoi, respectively (Yang et al. 2003). against Aedes aegypti and observed the LC50 to range from
The findings of our results is in corroboration with 60 to 533 ppm. Prajapati et al. (2005) reported that the
important findings of Sumroiphon et al. (2006) who have larvicidal activity of different plants essential oil showed
reported that the effect of water extract of citrus seed extract varied LC 95 values against Culex quinquefasciatus.
showed LC50 values of 135,319.40 and 127,411.88 ppm They were Pimpinella anism (149 μg/ml), Z. officinalis
against the larvae of Aedes aegypti and Culex quinquefascia- (202 μg/ml), Junipers macropoda (204 μg/ml), Cinnamo-
tus. Amer and Mehlhorn (2006a) have reported that the five mum zeylanicum (277 μg/ml), Curcuma longa (292 μg/ml),
most effective oils were those of Litsea (Litsea cubeba), Cyperus scariosus (408 μg/ml), Ocimum basilicum
Cajeput (Melaleuca leucadendron), Niaouli (Melaleuca quin- (315 μg/ml), Cuminum cyminum (344 μg/ml), and Nigella
quenervia), Violet (Viola odorata), and Catnip (Nepeta sativa (365 μg/ml). Rajkumar and Jebanesan (2004) studied
cataria), which induced a protection time of 8 h at the ovicidal activity of M. polystachyum leaf extract against
maximum and a 100% repellency against Aedes aegypti, Culex quinquefasciatus and observed 100% egg mortality
Anopheles stephensi, and Culex quinquefasciatus. The at 100 ml/l. Larvicidal activity of crude extract of Sida
essential oil of T. minuta providing a repellency of 90% acuta against Culex quinquefasciatus, Aedes aegypti, and
protection for 2 h against Anopheles stephensi, Culex Anopheles stephensi with LC50 values ranging between 38
quinquefasciatus, and Aedes aegypti was observed by Tyagi to 48 mg/l (Govindarajan 2010d); the lethal concentration
et al. (1994). Mullai and Jebanesan (2006) reported the (LC50) values of Ficus benghalensis against early second,
complete ovicidal activity (100% mortality) was attained at third, and fourth instar larvae of Culex quinquefasciatus,
300 ppm for methanol, benzene, petroleum ether, and ethyl Aedes aegypti, and Anopheles stephensi were 41.43, 58.21,
acetate extracts of Citrullus pubescens against Culex quin- and 74.32 ppm; 56.54, 70.29, and 80.85 ppm; and 60.44,
quefasciatus. The leaf extract of Solanum trilobatum reduced 76.41, and 89.55 ppm, respectively (Govindarajan 2010c).
egg laying by gravid females of Anopheles stephensi from Rahuman et al. (2008) have reported that the LC50 value of
18% to 99% compared with ethanol-treated controls at petroleum ether extracts of Jatropha curcas, Pedilanthus
0.01%, 0.025%, 0.05%, 0.075%, and 0.1% (Rajkumar and tithymaloides, Phyllanthus amarus, Euphorbia hirta, and
Jebanesan 2005). Bagavan et al. (2008b) have reported Euphorbia tirucalli were 8.79, 55.26, 90.92, 272.36, and
that peel chloroform extract of Citrus sinensis, leaf ethyl 4.25 ppm, respectively, against Aedes aegypti and 11.34,
acetate extracts of O. canum and O. sanctum, and leaf 76.61, 113.40, 424.94, and 5.52 ppm, respectively, against
chloroform extract of Rhinacanthus nasutus against the larvae Culex quinquefasciatus, and the larvicidal effect of ten
of Anopheles subpictus (LC50 =58.25, 88.15, 21.67, and plants corresponding to different botanical families on
40.46 ppm; LC90 =298.31, 528.70, 98.34, and 267.20 ppm) Anopheles stephensi and Culex quinquefasciatus. The
and peel methanol extract of Citrus sinensis, leaf methanol highest larval mortality was found in leaf acetone and
extract of O. canum, and ethyl acetate extracts of O. sanctum methanol of Canna indica (LC50 =29.62 and 40.77 ppm;
and R. nasutus against the larvae of Culex tritaeniorhynchus LC90 =148.55 and 165.00 ppm) against second instar larvae
(LC50 =38.15, 72.40, 109.12, and 39.32 ppm; LC90 =184.67, (LC 50 = 121.88 and 69.76, ppm; LC 90 = 624.35and
268.93, 646.62, and 176.39 ppm), respectively. Kamaraj et al. 304.27 ppm) and against fourth instar larvae of Anopheles
(2008b) reported that the highest larval mortality was found stephensi and in methanol and petroleum ether extracts of
in leaf petroleum ether, flower methanol extracts of Crypto- Ipomoea carnea (LC50 =41.82 and 39.32 ppm; LC90 =
coryne auriculata, flower methanol extracts of L. aspera and 423.76 and 176.39 ppm) against second instar larvae
R. nasutus, leaf and seed methanol extracts of Solanum (LC 50 = 163.81 and 41.75 ppm; LC 90 = 627.38 and
torvum, and leaf hexane extract of V. negundo against the 162.63 ppm) and against fourth instar larvae of Culex
larvae of Anopheles subpictus (LC50 =44.21, 44.69, 53.16, quinquefasciatus, respectively (Rahuman et al. 2009).
41.07, 35.32, 28.90, and 44.40 ppm; LC90 =187.31, 188.29, Govindarajan et al. (2008a) reported that the younger age
233.18, 142.66, 151.60, 121.05, and 192.11 ppm, respective- groups of egg rafts or eggs showed poor hatchability rate
ly) and against the larvae of Culex tritaeniorhynchus (LC50 = when exposed to higher concentrations of extract and that
69.83, 51.29, 81.24, 71.79, 44.42, 84.47, and 65.35 ppm; older age groups of egg rafts or eggs showed high
LC90 =335.26, 245.63, 300.45, 361.83, 185.09, 351.41, and hatchability rate when exposed to lower concentrations of
302.42 ppm, respectively). extract. The methanol containing water that served as
Ansari et al. (2005) observed the larvicidal activity of a control showed 94% hatchability in 0–3-h-old egg
Pinus longifolia oil against three vector mosquitoes namely rafts/eggs, but the 100% hatchability was noted in egg
364 Parasitol Res (2011) 109:353–367

rafts/eggs beyond the age of 0–3 h old in leaf methanol anguina, Luffa acutangula, Benincasa cerifera, and
(90%) extract of Cassia fistula against egg raft of Culex Citrullus vulgaris tested with LC50 values were 465.85,
quinquefasciatus (Govindarajan et al. 2008b). The benzene 567.81, 839.81, 1,189.30 and 1,636.04 ppm, respectively,
extracts of Citrullus vulgaris exerted 100% mortality (zero against the late third larval age group of Culex quinque-
hatchability) at 250 ppm, a very low hatchability (11.8%) at fasciatus (Prabakar and Jebanesan 2004). The leaf extract
200 ppm, and complete ovicidal activity at 300 ppm. The of Cassia fistula with different solvents, viz, methanol,
fraction I at 80 ppm exerted a very low hatchability rate of benzene, and acetone, was studied for the larvicidal,
3.2% followed by fraction II (6.9%), and fractions III and ovicidal, and repellent activity against Aedes aegypti. The
IV afforded 4.9% and 5.3% hatchability recorded against 24 h LC50 concentration of the extract against Aedes
Anopheles stephensi and Aedes aegypti, respectively aegypti were observed at 10.69, 18.27, and 23.95 mg/l,
(Mullai et al. 2008). The ovicidal effect of Solenostemma respectively (Govindarajan 2009).
argel was low; however, concentrations of 0.05% and 0.1% Mullai and Jebanesan (2007) have reported that ethyl
exhibited significant effects (p<0.05), producing 65% and acetate, petroleum ether, and methanol leaf extracts of
75% and 62.9% and 62.9%, respectively, on the 1st and 2nd Citrullus colocynthis and Cucurbita maxima showed LC50
day after treatment, respectively. The 0.1% concentration values of 47.58, 66.92, and 118.74 ppm and 75.91, 117.73,
reduced egg hatch by 33.7%, compared with the control, and 171.64 ppm, respectively, against Culex quinquefascia-
and 100% mortality values were evident in concentrations tus larvae. Komalamisra et al. (2005) have reported that the
as low as 0.025% at 2 days posthatching against Culex petroleum ether and methanol (MeOH) extracts of R.
pipiens (Al-Doghairi et al. 2004). The seed extract of nasutus and Derris elliptica exhibited larvicidal effects
Atriplex canescens showed complete ovicidal at 1,000 ppm against Aedes aegypti, Culex quinquefasciatus, Anopheles
concentration in eggs of Culex quinquefasciatus (Ouda et dirus, and Mansonia uniformis with LC50 values between
al. 1998). The bioactive compound Azadirachtin isolated 3.9 and 11.5 mg/L, while the MeOH extract gave LC50
from Azadirachta indica showed complete ovicidal activity values of between 8.1 and 14.7 mg/L. D. elliptica
in eggs of Culex tarsalis and Culex quinquefasciatus petroleum ether extract showed LC50 values of between
exposed to 10 ppm concentration (Ouda et al. 1998). 11.2 and 18.84 mg/L, and the MeOH extract exhibited LC50
Earlier authors reported that the methanol leaf extracts of values between 13.2 and 45.2 mg/L. Earlier authors
V. negundo, V. trifolia, V. peduncularis, and V. altissima reported that the n-hexane, ethyl acetate, and methanol
were used for larvicidal assay with LC50 value of 212.57, extracts of Cassia nigricans showed 100% larval mortality
41.41, 76.28, and 128.04 ppm, respectively, against the against Ochlerotatus triseriatus (Georges et al. 2008). Jang
early fourth instar larvae of Culex quinquefasciatus et al. (2002) have reported that the methanol extracts of
(Kannathasan et al. 2007). The same extracts of Euphorbia Cassia obtusifolia, Cassia tora, and Vicia tetrasperma
tirucalli latex and stem bark were evaluated for larvicidal exhibited more than 90% larval mortality at 200 ppm on
activity against laboratory-reared larvae of Culex quinque- Aedes aegypti and Culex pipiens. The larvicidal activity of
fasciatus with LC50 values of 177.14 and 513.387 mg/l, petroleum ether, ethanolic, aqueous extracts of dried leaves,
respectively (Yadav et al. 2002). Sharma et al. (2005) and fixed oil from the seeds of Caesalpinia bonduc
reported that the acetone extract of Nerium indicum and (Family: Caesalpiniaceae) showed 100% mortality in 1%
Thuja orientelis have been studied with LC50 values of concentration of petroleum ether and ethanolic extract of
200.87, 127.53, 209.00, and 155.97 ppm against third instar leaf, whereas it was 55% in 2.5% concentration of aqueous
larvae of Anopheles stephensi and Culex quinquefasciatus. extract and 92.6% in 2.5% concentration of fixed oil against
Larvicidal activity of the same extracts of Murraya the fourth instar larvae of Culex quinquefasciatus (Saravanan
koenigii, Coriandrum sativum, Ferula asafoetida, and et al. 2007); the petroleum ether extract of Solanum
Trigonella foenum graceum was tested out using different xanthocarpum was observed to be the most toxic with
concentrations of each plant (range, 25–900 ppm) against LC50 of 1.41 and 0.93 ppm and LC90 of 16.94 and 8.48 ppm
Aedes aegypti larvae (Harve and Kamath 2004); Rahuman at 24 and 48 h after application, respectively, against
and Venkatesan (2008) reported that the petroleum ether Anopheles stephensi (Mohan et al. 2007). Tawatsin et al.
extract of Citrullus colocynthis; methanol extracts of (2001) have reported repellent activity against Aedes aegypti,
Cannabis indica, Cannabis sativus, and Momordica char- Anopheles dirus, and Culex quinquefasciatus, which is due
antia; and acetone extract of Trichosanthes anguina against to 5% vanillin, which has been added to the essential oil of
the larvae of Aedes aegypti (LC50 =74.57, 309.46, 492.73, Curcuma longa. Venketachalam and Jebanesan (2001) have
199.14, and 554.20 ppm) and against Culex quinquefascia- also reported that the repellent activity of methanol extract of
tus (LC 5 0 = 88.24, 377.69, 623.80, 207.61, and Ferronia elephantum leaves against Aedes aegypti activity at
842.34 ppm), respectively. Larvicidal efficacies of metha- 1.0 and 2.5 mg/cm2 concentrations gave 100% protection up
nol extracts of Momordica charantia, Trichosanthes to 2.14±0.16 h and 4.00±0.24 h, respectively, and the total
Parasitol Res (2011) 109:353–367 365

percentage protection was 45.8% at 1.0 mg/cm2 and 59.0% Bowers WS, Sener B, Evans PH, Bingol F, Erdogan I (1995) Activity
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and Anopheles gambiae. Insect Sci Appl 16(3/4):339–342
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that the experimental plant extracts were effective to control Larvicidal activity of essential oils from Brazilian plants against
Anopheles stephensi, Aedes aegypti, and Culex quinquefas- Aedes aegypti L. Mem Inst Oswaldo Cruz 99:541–544
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Acknowledgments The authors are grateful to the Dr. (Mrs.) Govindarajan M (2010b) Evaluation of indigenous plant extracts
Selvi Sabhanayakam, Professor and Head, Department of Zoology, against the malarial vector, Anopheles stephensi (Liston) (Diptera:
Annamalai University, for the laboratory facilities provided. We Culicidae). Parasitol Res. doi:10.1007/s00436-010-2224-0
acknowledge the staff members of the VCRC (ICMR), Pondicherry Govindarajan M (2010c) Larvicidal efficacy of Ficus benghalensis L.
for their cooperation. plant leaf extracts against Culex quinquefasciatus Say, Aedes
aegypti L. and Anopheles stephensi L. (Diptera: Culicidae). Eur
Rev Med Pharmacol Sci 14:107–111
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