Larvicidal efficacy of latex and extract of Calotropis procera (Gentianales: Asclepiadaceae) against Culex quinquefasciatus and Anopheles stephensi (Diptera: Culicidae)

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J Vector Borne Dis 47, September 2010, pp. 185–188
Larvicidal efficacy of latex and extract of Calotropis procera
(Gentianales: Asclepiadaceae) against Culex quinquefasciatus and
Anopheles stephensi (Diptera: Culicidae)
M. Shahia, A.A. Hanafi-Bojdb, M. Iranshahic, H. Vatandoostb & M.Y. Hanafi-Bojdd
aSchool of Public Health, Hormozgan University of Medical Sciences, Bandar Abbas; bDepartment of Medical Entomology &
Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran; cDepartment of Pharmacognosy,
Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad; dDepartment of
Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Key words Anopheles stephensi; Calotropis procera; Culex quinquefasciatus; larvicidal activity
Mosquitoes are responsible for transmission of many
diseases to human. They not only can carry diseases
that afflict humans, but also transmit several diseases
and parasites to birds, dogs, horses, etc. Anopheles
stephensi and Culex quinquefasciatus (Diptera: Cu-
licidae) are urban and rural mosquito pests in Iran.
Hormozgan province, southern Iran, has a favourable
climate for mosquito breeding and development, with
active foci of malaria transmission. Extensive appli-
cation of chemical insecticides for many years in this
area caused irrepairable damages to environment and
resulted in resistance in An. stephensi to some insec-
ticides/larvicides1,2.
Several botanicals offer great promise as sources of
phytochemicals for the control of mosquitoes. One
of the earliest reports of the use of plant extracts
against mosquito larvae is extraction of plant alka-
loids like nicotine, anabasine, methyl anabasine and
lupinine from the Russian weed in 1933. Anabasis
aphylla killed larvae of Culex pipiens Linn., Cx.
territans Walker, and Cx. quinquefasciatus Say3. Six
plant families with several representative species,
Asteraceae, Cladophoraceae, Labiatae, Meliaceae,
Oocystaceae and Rutaceae, appear to have the great-
est potential for providing future mosquito control
agents. Sukumar et al3 has reviewed botanical de-
rivatives in mosquito control and also discussed about
botanical phytochemicals with mosquitocidal poten-
tial, using examples of different studies4.
Some publications have described antibacterial, an-
algesic or schizontocidal activities for the latex of
Calotropis procera5–8. Giridhar et al9 found com-
pounds with larvicidal activity in the latex of C.
procera for the first time. Extracts of C. procera have
insecticidal activity against different insects such
as Sarcophaga haemorrhoidalis10, while the latex
was used against the third stage larvae of Musca
domestica11. The crude latex produced by the green
parts of the plant was evaluated for its toxic effects
upon egg hatching and larval development12. They
found the whole latex was shown to cause 100%
mortality of III instars within 5 min. Different aque-
ous concentrations of this plant affected the gravid
female Aedes aegypti mosquitoes and this behaviour
continued till three gonotrophic cycles13.
The aim of this survey was to find the larvicidal effi-
cacy of native C. procera of southern Iran against
An. stephensi, the main malaria vector of this area1
and Cx. quinquefasciatus, a potential vector of some
arboviral infections as well as filariasis.
Calotropis procera or milkweed naturally grows in
southern parts of Iran. The fresh leaves of this plant
were collected from Bandar Abbas County, an en-
demic area of malaria in southern Iran14, bordered
with Persian Gulf. Fresh leaves of C. procera be-
came dried in the room temperature far from direct
sunlight. Methanolic extracts were obtained with

J VECTOR BORNE DIS 47, SEPTEMBER 2010
186
maceration method in the School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad,
Iran. Latex was collected by cutting the fresh leaves
using a razor blade and gathered into glass vials. Lar-
vae were obtained from two established colonies of
An. stephensi and Cx. quinquefasciatus in the insec-
tary of Bandar Abbas School of Public Health,
Hormozgan University of Medical Sciences. Larvi-
cidal tests were perforemd based on WHO standard
method15.
Tests were conducted using concentrations of 4, 8,
16, 32, 64, 128, 256, 512 and 1024 ppm of extract,
and 4, 8, 16, 32, 64, 128, 256 ppm of plant latex
against the larvae of the above mentioned two spe-
cies of mosquitoes. Tests were conducted using four
batches of 25 larvae (a total of 100 larvae) and one
batch as untreated control group for each concen-
tration. Tested larvae were maintained under the in-
sectary conditions (Temperature 27–29°C, relative
humidity 70–75% and 12 : 12 light : dark period).
Mortality rates were observed and recorded after
24 h. Tests with untreated control group mortality
between 5 and 20% were corrected by Abbott’s for-
mula16, meanwhile above 20% were repeated. The
LC50 and LC90 values were obtained by Probit Analy-
sis17. Toxic activity was reported as LC50 and LC90
representing the concentrations that induced 50 and
90% mortality after 24 h.
The results for the toxicity of milkweed derivatives
against An. stephensi and Cx. quinquefasciatus lar-
vae are presented in Table 1. The alcoholic extract
of C. procera showed to be less toxic than latex in
both mosquito species. The LC50 values were 109.71
and 387.93 mg/l for An. stephensi and Cx.
quinquefasciatus, respectively. These figures were
13.06 and 86.47 mg/l respectively for latex of the
plant. Overall, An. stephensi larvae were more sus-
ceptible than Cx. quinquefasciatus to both latex and
extract. The 512 ppm concentration of plant extract
didn’t show a mortality rate >78% in Cx.
quinquefasciatus after 24 h. But in the case of An.
stephensi we observed >95% mortality after 24 h
from 256 ppm. Tests with latex showed 99% mor-
tality at 64 ppm for An. stephensi, only 44% mortal-
ity against Cx. quinquefasciatus and a maximum of
67% in 256 ppm.
Botanical derivatives may be the future of mosquito
control programs. Phytochemicals that are tested
against various life stages of mosquitoes, have po-
tential uses as growth and reproduction inhibitors,
repellents and as oviposition deterrents. In the past
decades, studies on the effect of plant extracts on
mosquito vectors of disease indicated that these ma-
terials are good alternatives for synthetic chemical
pesticides. These studies have determined that some
botanical compounds such as alkaloids, nicotine,
anabasin and lupitin in the extracts produced high
mortality against mosquito larvae. Mosquito species,
life stage, the plant parts and solvent used for ex-
traction, phototoxic activity and the geographical
origin of a plant compound are important factors in
the efficacy of a phytochemical3,4.
Study of Ramos et al12 showed that the C. procera
latex contains the larvicidal compounds, which caused
100% mortality in III stage larvae of Ae. aegypti af-
ter 5 min. Study of Markouk et al18 showed that C.
procera latex caused 50% mortality against An.
labranchiae at 28 ppm. The results of our survey
Table 1. Larvicidal activity of alcoholic extract and fresh latex of Calotropis procera against
An. stephensi and Cx. quinquefasciatus larvae in Iran, 2008
Botanical Species LC50 (Confidence limits) LC90 (Confidence limits)
Alcoholic extract An. stephensi 109.71 (99.37–120.54) 234.61 (206.68–275.46)
Cx. quinquefasciatus 387.93 (359.66–418.69) 630.66 (566.54–731.41)
Fresh latex An. stephensi 13.06 (9.74–21.81) 23.53 (16.16–85.36)
Cx. quinquefasciatus 86.47 (70.44–143.89) 973.89 (613.88–1812.05)

187
SHAHI ET AL: MOSQUITO LARVICIDAL ACTIVITY OF CALOTROPIS PROCERA
confirmed the efficacy of latex and extract compo-
nents of C. procera and mortality increased with con-
centration. Also we found latex a strong killer against
both Cx. quinquefasciatus and An. stephensi larvae
compared to extract (Table 1). Considering the proof
of some pesticide compounds such as Calotropin and
Calotoxin in the milkweed plant extracts19 it seems
they are the most important factors in causing mor-
tality of mosquito larvae. In the same concentrations,
the fatality rate caused by latex is more than the al-
coholic extract. It can be due to obtaining the plant
extracts from dried parts like leaves, so they will loss
some effective components that exist in fresh latex,
during the drying process and therefore will induce
lower mortality rates in mosquito larvae. In regard
to availability of C. procera in southern parts of Iran,
it is suggested to perform more studies to produce
natural insecticide/larvicide from this native plant.
The latex of C. procera has shown larvicidal effi-
cacy against all three important vector species: Ae.
aegypti, An. stephensi and Cx. quinquefasciatus in
India20. They used 14 different solvents to dissolve
the latex at 1000 ppm and found methanolic extract
produced 100% mortality in Ae. aegypti larvae. Their
results showed that Cx. quinquefasciatus is more
susceptible than An. stephensi at same concentra-
tions of latex. This is in contrary with our results.
Study on larvicidal efficacy of C. procera by Markouk
et al18 which obtained a LC50 of 28 mg/l with aque-
ous latex of this plant against An. labranchiae, while
the ethanolic extract of root showed LC50 of 315
mg/l against this Anopheles. In the case of An.
stephensi Shaalan et al4 reported 12 plant species
tested for larvicidal effects. The values are more than
those obtaind in the present study. The LC50 values
ranged from 3.9 mg/l for seed fractions of
Calophyllum inophyllum to 1000 mg/l for ethanolic
extract of leaf of Cannabis sativa.
Larvicidal activity of Iranian plants Tagetes minuta
L. and Cymbopogon olivieri (Boiss.) Bar against An.
stephensi larvae are studied under the laboratory
conditions21–23. The results of these studies showed
the essential oil of Tagetes minuta L. is stronger lar-
vicide against An. stephensi, rather than its methanolic
extract; while the essential oil of Cymbopogon olivieri
(Boiss.) Bar has interesting activity against larvae of
An. stephensi. The only field examination of plant
derived larvicides is conducted by Vatandoost and
Vaziri24, who reported LC50 and LC90 values of 0.35
and 1.81 mg/l for An. stephensi, and 0.69 and 3.18
mg/l for Cx. quinquefasciatus respectively using
Neemarin, a plant derived larvicide which obtains
from Azadirachta indica.
From the results, it can be concluded that C. procera
extracts possess good larvicidal activity against mos-
quitoes and more studies are indicated to extract the
active compounds for future studies and use in mos-
quito control.
Acknowledgement
The authors wish to thank Eng. R. Jafari, Isfahan
Health Research Station, for his assistance to pre-
pare some references and Mr H. Javdan for labora-
tory rearing the mosquitoes for this survey. This
project is funded by Research Deputy, Hormozgan
University of Medical Sciences, Bandar Abbas, Iran.
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Corresponding author: Dr A.A. Hanafi-Bojd, Department of Medical Entomology & Vector Control, School of Public Health,
Tehran University of Medical Sciences, Tehran, Iran.
E-mail: aahanafibojd@tums.ac.ir
Received: 20 October 2009 Accepted in revised form: 15 May 2010