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ORIGINAL PAPER
Mosquito larvicidal, pupicidal, adulticidal, and repellent
activity of Artemisia nilagirica (Family: Compositae) against
Anopheles stephensi and Aedes aegypti
Chellasamy Panneerselvam &Kadarkarai Murugan &
Kalimuthu Kovendan &Palanisamy Mahesh Kumar
Received: 20 July 2012 /Accepted: 1 August 2012
#Springer-Verlag 2012
Abstract Mosquito-borne diseases have an economic im-
pact, including loss in commercial and labor outputs, particu-
larly in countries with tropical and subtropical climates;
however, no part of the world is free from vector-borne dis-
eases. The aim of the present study, to evaluate the larvicidal,
pupicidal, repellent, and adulticidal activities of methanol
crude extract of Artemisia nilagirica were assayed for their
toxicity against two important vector mosquitoes, viz.,
Anopheles stephensi and Aedes aegypti (Diptera: Culicidae).
The fresh leaves of A. nilagirica were washed thoroughly in
tap water and shade dried at room temperature (28± 2 °C) for
5–8 days. The air-dried materials were powdered separately
using commercial electrical blender. From the plants, 500 g
powdered was macerated with 1.5 L organic solvents of
methanol sequentially for a period of 72 h each and filtered.
The larval and pupal mortality was observed after 24 h of
exposure; no mortality was observed in the control group. The
first- to fourth-instar larvae and pupae of A. stephensi had
values of LC
50
0272.50, 311.40, 361.51, 442.51, and
477.23 ppm, and the LC
90
0590.07, 688.81, 789.34, 901.59,
and 959.30 ppm; the A. aegypti had values of LC
50
0300.84,
338.79, 394.69, 470.74, and 542.11 ppm, and the LC
90
0
646.67, 726.07, 805.49, 892.01, and 991.29 ppm, respective-
ly. The results of the repellent activity of plant extract of A.
nilagirica plants at five different concentrations of 50, 150,
250, 350, and 450 ppm were applied on skin of fore arm in
man and exposed against adult female mosquitoes. In this
observation, the plant crude extract gave protection against
mosquito bites without any allergic reaction to the test person,
and also, the repellent activity is dependent on the strength of
the plant extracts. The adult mortality was found in methanol
extract of A. nilagirica,withtheLC
50
and LC
90
values of
205.78 and 459.51 ppm for A. stephensi, and 242.52 and
523.73 ppm for A. aegypti, respectively. This result suggests
that the leaf extract have the potential to be used as an ideal
eco-friendly approach for the control of vector mosquito as
target species.
Introduction
Mosquitoes are the principal vector of many vector-borne
diseases affecting human beings and animals, in addition to
nuisance. Vector-borne diseases in India, e.g., malaria, den-
gue, chikungunya, filariasis, Japanese encephalitis, and leish-
maniasis, cause thousands of deaths per year. India reports
1.48 million malarial cases and about 1,173 deaths, 1.4 mil-
lion suspected and 1,985 confirmed chikungunya cases, 5,000
Japanese encephalitis cases and approximately 1,000 deaths,
and 383 dengue cases and 6 deaths during 2006 and 2007
(WHO 2007;GopalanandDas2009;Dhimanetal.2010).
Anopheles stephensi is the primary vector of malaria in
India and other West Asian countries, and improved methods
of control are urgently needed (Burfield and Reekie 2005;
Mittal et al. 2005). Malaria infects more than 500 million
humans each year, killing approximately 1.2–2.7 million per
year. About 90 % of all malaria cases occur in Africa, as does
approximately 90 % of the world’s malaria-related deaths
(Breman et al. 2004). Malaria, caused by Plasmodium falci-
parum, is one of the leading causes of human morbidity and
mortality from infectious diseases, predominantly in tropical
and subtropical countries (Snow et al. 2005). Mosquito bites
may also cause allergic responses including local skin reac-
tions and systemic reactions such as urticaria and angioedema
(Peng et al. 2004). Botanical and microbial insecticides have
been increasingly used for mosquito control because of their
C. Panneerselvam :K. Murugan :K. Kovendan (*):
P. Mahesh Kumar
Division of Entomology, Department of Zoology,
School of Life Sciences, Bharathiar University,
Coimbatore 641 046, Tamil Nadu, India
e-mail: gokulloyo@yahoo.co.in
Parasitol Res
DOI 10.1007/s00436-012-3073-9
efficacy and documented nontoxic effects on nontarget organ-
isms (Ascher et al. 1995). The highest number of malaria,
P. falciparum cases and malaria-related deaths are recorded
from the state of Orissa located in the eastern part of India
(Sharma et al. 2010).
Aedes aegypti is responsible for spreading dengue and
chikungunya. Dengue is prevalent throughout the tropics
and subtropics. The World Health Organization estimates that
around 2.5 billion people are at risk of dengue. Infections have
dramatically increased in recent decades due to increased
urbanization, trade, and travel. No effective drug or vaccine
is available so far. The only solution is to prevent the disease-
carrying mosquito from breeding and biting humans. Dengue
is the most important mosquito spread viral disease and a
major international public health concern. It is a self-limiting
disease found in tropical and subtropical regions around the
world, predominantly in urban and semiurban areas. Dengue
fever or dengue hemorrhagic fever is caused by dengue virus,
which belongs to genus Flavivirus and family Flaviviridae,
and includes serotypes 1, 2, 3, and 4 (Den-1, Den-2, Den-3,
and Den-4) (WHO 2010).
Botanical phytochemicals with mosquitocidal potential
are now recognized as potent alternative insecticides to
replace synthetic insecticides in mosquito control programs
due to their excellent larvicidal, pupicidal, and adulticidal
properties. Many synthetic insecticides and naturally occur-
ring chemical cues have been shown to influence mosquito
oviposition (Millar et al. 1992; Olagbemiro et al. 1999;
Geetha et al. 2003). The chemicals derived from plants have
been projected as weapons in future mosquito control pro-
gram as they are shown to function as general toxicant,
growth and reproductive inhibitors, repellents and
oviposition-deterrent (Sukumar et al. 1991). Essential oil
of Cinnamomum zeylanicum showed oviposition-deterrent
and repellent activities, and the essential oils of Zingiber
officinale and Rosmarinus officinalis also showed both ovi-
cidal and repellent activities against A. stephensi,A. aegypti,
and C. quinquefasciatus (Prajapati et al. 2005). Barnard
(1999) tested the repellency of five essential oils (Bourbon
geranium, cedarwood, clove, peppermint, and thyme) singly
applied at different concentrations or in combinations
against two mosquito species A. aegypti and A. albimanus.
Karunamoorthi et al. (2008) have reported that the petro-
leum ether (60–80 °C) extracts of the leaves of Vitex
negundo were evaluated for larvicidal activity against larval
stages of C. tritaeniorhynchus. The toxicity of dichlorome-
thane, petroleum ether, and methanol extracts from V.
negundo seed and leaf to the second- and fourth-instar
larvae showed oviposition-deterrent effects on Plutella
xylostella (Yuan et al. 2006), and the oil obtained from
leaves was evaluated against A. aegypti (Hebbalkar et al.
1992). Samidurai et al. (2009) observed that the leaf extracts
of Pemphis acidula were evaluated for larvicidal, ovicidal,
and repellent activities against C. quinquefasciatus and A.
aegypti. Mullai et al. (2008) have reported that the leaf
extract of Citrullus vulgaris with different solvents, viz.,
benzene, petroleum ether, ethyl acetate, and methanol, was
tested for larvicidal, ovicidal, repellent, and insect growth
regulatory activities against A. stephensi.
Artemisia nilagirica (C.B. Clarke) Pampan. (Makkipu in
Tamil) is a valuable medicinal plant of compositae included
in threatened category. It belongs to family Compositae
(Fig. 1). The bioactive compounds like volatile oils, sesqui-
terpene lactones, and flavonoids reported in the species are
having insecticidal, antimicrobial, and antiparastical proper-
ties (Borzabad et al. 2010). In traditional medicine, this
plant is being widely used for the treatment of diabetes,
epilepsy, depression, insomonia, and anxiety stress (Walter
et al. 2003). All the parts of the plant are used as antihel-
mintic, antiseptic, antispasmodic, carminative, cholagogue,
digestive, expectorant, purgative, and stimulant also. The
essential oils of the plant were reported to exhibit 90 %
mosquito repellency against the mosquito A. aegypti that
transmits yellow fever (Ram and Mehrotra 1995). A paste or
powder form of the leaves is applied for the treatment of
skin diseases (Kapoor 2000). Due to the overexploitation for
its medicinal uses, the species become lower in population
size in high altitudes of Nilgiris (Paulsamy et al. 2008).
Taxonomy
Kingdom: Plantae
Subkingdom: Viridaeplantae
Phylum: Tracheophyta
Subphylum: Euphyllophytina
Class: Spermatopsida
Subclass: Asteridae
Superorder: Campanulanae
Order: Asterales
Family: Compositae
Subfamily: Asteroideae
Fig. 1 Artemisia nilagirica
Parasitol Res
Tribe: Anthemideae
Genus: Artemisia
Species: nilagirica (C.B.Clarke) Pamp.
Botanical name: Artemisia nilagirica Leila M. Shultz
(2012)
It is the aromatic shrub found throughout the mountains
districts of India, often gregarious, pubescent or villous
throughout; lower leaves ovate in outline deeply pinnatisect
with small stipule-like lobes at the base, pubescent above,
white tomentose beneath, upper most smaller, 3-fid or en-
tire, lanceolate; panicled racemer, outer flowers female, very
slender, inner disk flowers fertile, bisexual, bracts ovate, or
oblong, margins scarious fruits oblong ellipsoid minute
achene’s (Chopra et al. 1980). The plant contains sesquiter-
pene lactones, exiguaflavone A and B, macckianin, and 2-
(2,4-dihydroxyphenyl)-5,6-methylenedioxy benzofuran
(Kirtikar and Basu 1975). It is also said to be anthelmintic,
antiseptic, and expectorant, astringent, aromatic, anti-
inflammatory, appetizer, digestive, and diuretic. It is also
used in cough, asthma, leprosy skin disease, and as antisep-
tic (Yodhathai and Sombat 2002; Shafi et al. 2004; Ganguly
et al. 2006). Larvicidal activity was found ever since the
discovery of the larvicidal potential of the petroleum ether
extract of Artemisia nilagirica and Galinsoga quadriradiata
(Sakthivadivel and Daniel 2008) and dichloromethane
whole plant extracts of Citrullus colocynthis against C.
quinquefasciatus (Arivoli and Samuel 2011).
The methanol extracts of Pelargonium citrosa leaf were
tested for their biological, larvicidal, pupicidal, adulticidal,
antiovipositional activity, repellency, and biting deterrency
against A. stephensi (Jeyabalan et al. 2003). The ethanol
extract of Curcuma aeruginosa,Curcuma aromatica, and
Curcuma xanthorrhiza were tested for repellent activity
against A. togoi,Armigeres subalbatus,C. quinquefasciatus,
and C. tritaeniorhynchus (Pitasawat et al. 2003). Many
researchers have reported on the effectiveness of plant extract
against mosquito larvae (Kalyanasundaram and Das 1985;
Mahesh Kumar et al. 2012a; Kovendan and Murugan 2011;
Kovendan et al. 2012c,d,e,g).
In view of an increasing interest in developing plant
origin insecticides as an alternative to chemical insecticides,
this study was undertaken to assess the larvicidal, pupicidal,
adulticidal, and repellent activities of the leaf extract from A.
nilagirica against malarial vector, A. stephensi and dengue
vector, A. aegypti as target species.
Materials and methods
Collection of eggs and maintenance of larvae
The eggs of A. stephensi and A. aegypti were collected from
the National Centre for Disease Control field station of
Mettupalayam, Tamil Nadu, India, using an “O”-type brush.
These eggs were brought to the laboratory and transferred to
18×13×4-cm enamel trays containing 500 mL of water for
hatching. The mosquito larvae were pedigree dog biscuits
and yeast at 3:1 ratio. The feeding was continued until the
larvae transformed into the pupal stage.
Maintenance of pupae and adults
The pupae were collected from the culture trays and trans-
ferred to plastic containers (12 × 12 cm) containing 500 mL
of water with the help of a dipper. The plastic jars were kept
in a 90×90× 90-cm mosquito cage for adult emergence.
Mosquito larvae were maintained at 27 + 2 °C, 75–85 %
relative humidity, under a photoperiod of 14:10 (light/dark).
A 10 % sugar solution was provided for a period of 3 days
before blood feeding.
Blood feeding of adult mosquito vectors
The adult female mosquitoes were allowed to feed on the
blood of a rabbit (a rabbit per day, exposed on the dorsal
side) for 2 days to ensure adequate blood feeding for
5 days. After blood feeding, enamel trays with water
from the culture trays were placed in the cage as ovi-
position substrates.
Collection of plants and preparation of plant extracts
The A. nilagirica plants were collected in and around
Nilgiris, Western Ghats, Coimbatore, Tamil Nadu, India.
The plants were identified by the Taxonomist, Department
of Botany, Bharathiar University, Coimbatore. The fresh
leaves of A. nilagirica were washed thoroughly in tap water
and shade dried at room temperature (28 ±2 °C) for 5–8 days.
The air-dried materials were powdered separately using
commercial electrical blender. From the plants, 500 g pow-
dered was macerated with 1.5 L organic solvents of metha-
nol sequentially for a period of 72 h each and filtered. The
yield of the A. nilagirica crude extract by methanol 15.23 g,
respectively. The extracts were concentrated at reduced tem-
perature on a rotary vacuum evaporator and stored at a
temperature of 4 °C. One gram of the plant residue was
dissolved in 100 mL of acetone (stock solution) considered
as 1 % stock solution. From this stock solution, concentra-
tions were prepared ranging from 180, 260, 340, 420, and
500 ppm, respectively.
Larval and pupal toxicity test
Laboratory colonies of mosquito larvae/pupae were used for
the larvicidal/pupicidal activity. Twenty-five numbers of
first to fourth instars larvae and pupae were introduce into
Parasitol Res
500-mL glass beaker containing 249 mL of dechlorinated
water, and 1 mL of desired concentrations of plant extract
was added.Larval food was given for the test larvae. At
each tested concentration two to five trials were made and
each trial consisted of five replicates. The control was setup
by mixing 1 mL of acetone with 249 mL of dechlori-
nated water. The larvae and pupae were exposed to
dechlorinated water without acetone served as control.
The control mortalities were corrected using Abbott’s
formula (Abbott’s1925).
Corrected mortality ¼Observed mortality in treatment Observed mortality in control
100 Control mortality 100
Percentage mortality ¼Number of dead larvae pupae
=
Number of larvae pupae introduced
=100
The LC
50
and LC
90
were calculated from toxicity data by
using probit analysis (Finney 1971).
Repellent bioassay
The stock solutions of the extracts were diluted with ace-
tone, polysorbate 80, and distilled water to obtain test sol-
utions of 50, 150, 250, 350.00, and 450 ppm. For repellent
experiment, 50 laboratory-reared bloods starved adult fe-
male mosquitoes that were between 3 and 10 days old were
placed into separate laboratory cages (45×45×40 cm).
Before each test, the forearm and hand of a human subject
were washed with unscented neutral soap, thoroughly
rinsed, and allowed to dry 10 min before extracts applica-
tion. The different plant extracts being tested were applied
from the elbow to the fingertips. The arm was left undis-
turbed. An arm treated with acetone and polysorbate 80
served as control. The control and treated arms were intro-
duced simultaneously into the cage. The numbers of bites
were counted over 5 min, every 30 min, from 1800 to 0600
hours. Protection time was recorded as the time elapsed
between repellent application and the observation period
immediately preceding that in which a confirmed bite was
obtained. If no bites were confirmed at 180 min, tests were
discontinued, and protection time was recorded as 180 min.
An attempt of the mosquito to insert its stylets was consid-
ered a bite. No mosquito attempted to bite the control arm
during the observation period; that trial was discarded, and
the test was repeated with a new batch of mosquitoes to
ensure that lack of bites was due to repellence and not to
mosquitoes not being predisposed to get a blood meal at the
time. The experiments were conducted five times in separate
cages, and in each replicate, different volunteers were used
to nullify any effect of skin differences on repellency. It was
observed that there was no skin irritation from the plant
extract. The percentage protection was calculated using the
following formula (Fradin and Day 2002; Venkatachalam
and Jebanesan 2001).
Protection ¼No:of bites received by control armðÞNo:of bites received by treated armðÞ½
No:of bites received by control armðÞ
100
Adulticidal bioassay
Sugar-fed adult female mosquitoes (5–6daysold)were
used. The A. nilagirica leaf extract were diluted with ace-
tone to make different concentrations. The diluted plant
extracts were impregnated on filter papers (140 × 120 mm).
A blank paper consisting of only ethanol was used as
control. The papers were left to dry at room temperature to
evaporate off the ethanol overnight. Impregnated papers
were prepared fresh prior to testing. The bioassay was con-
ducted in an experimental kit consisting of two cylindrical
plastic tubes both measuring 125×44 mm following the
method in WHO (1981). One tube served to expose the
mosquitoes to the plant extract, and another tube was used
to hold the mosquitoes before and after the exposure peri-
ods. The impregnated papers were rolled and placed in the
exposure tube. Each tube was closed at one end with a 16
mesh size wire screen. Sucrose-fed and blood starved mos-
quitoes (20) were released into the tube, and the mortality
effects of the extracts were observed every 10 min for 3 h
exposure period. At the end of 1, 2, and 3 h exposure
periods, the mosquitoes were placed in the holding tube.
Cotton pads soaked in 10 % sugar solution with vitamin B
complex was placed in the tube during the holding period of
Parasitol Res
24 h. Mortality of the mosquitoes was recorded after 24 h.
The above procedure was carried out in triplicate for plant
extract of each concentration.
Statistical analysis
The average adult mortality data were subjected to probit
analysis for calculating LC
50
,LC
90
, and other statistics at
95 % fiducidal limits of upper fiducidal limit and lower
fiducidal limit, and chi-square values were calculated by
using the SPSS Statistical software package 16.0 version
was used. Results with P<0.05 were considered to be sta-
tistically significant.
Results
Larvicidal and pupicidal activity of methanol leaf extract of
A. nilagirica at various concentrations against the malarial
vector, A. stephensi is given in the Table 1. Considerable
mortality was evident after the treatment of A. nilagirica for
all larval instars and pupae. Mortality was increased as the
concentration increased, for example, in first instars stage at
200 ppm concentration the larval mortality was 41 %,
whereas at 600 ppm concentration, it was increased to
94 %. In pupal mortality at 200 ppm concentration, it was
23 % increased to 63 % at 600 ppm (Fig. 2). The LC
50
and
LC
90
values were represented as follows: LC
50
value of first
instar was 272.50 ppm, second instar was 311.40 ppm, third
instar was 361.51 ppm, and fourth instar was 442.51 ppm,
respectively. LC
90
value of first instar was 590.07 ppm,
second instar was 688.81 ppm, third instar was 789.34 ppm,
and fourth instar was 901.59 ppm, respectively. The LC
50
value pupae was 477.23 ppm, and LC
90
value pupae was
959.30 ppm, respectively.
Larval and pupal mortality of A. aegypti after the treat-
ment of methanol extract of A. nilagirica was observed.
Table 2provides the larval and pupal mortality of A. aegypti
(first to fourth instars) after the treatment of A. aegypti at
different concentrations (200–600 ppm). Thirty-eight per-
cent mortality was noted at first instars larvae by the treat-
ment of A. nilagirica at 200 ppm, whereas it has been
increased to 89 % at 600 ppm of A. nilagirica extract
treatment (Fig. 2). Similar trend has been noted for all the
instars of A. aegypti at different concentration of A. nilagir-
ica treatment. The LC
50
and LC
90
values were represented
as follows: LC
50
value of first instar was 300.84 ppm, that of
second instar was 338.79 ppm, that of third instar was
394.69 ppm, and that of fourth instar was 470.74 ppm,
respectively. The LC
50
value of pupae was 542.11 ppm,
and the LC
90
value of pupae was 991.29 ppm, respectively.
The 95 % confidence limits LC
50
and LC
90
(LFL–UFL)
were also calculated. The results of larvicidal activity clearly
indicate that the percentage of mortality being directly pro-
portional to the concentration of the extract. This proves that
concentration plays important role in larvicidal activity. Chi-
square value was significant at P<0.05 level. Each test
included a control group with five replicates for each indi-
vidual concentration.
In the present observation, the results from the skin
repellent activity of methanol extract of A. nilagirica against
blood starved adult female of A. aegypti and A. stephensi are
given in Table 3. The methanol extracts of A. nilagirica
show significant repellency against A. aegypti and A. ste-
phensi. In this observation, this plant crude extract gave
protection against mosquito bites without any allergic reac-
tion to the test person, and also, the repellent activity is
dependent on the strength of the plant extract. The highest
repellency of 180 min was observed in methanol extract of
A. nilagirica against A. stephensi followed by A. aegypti,
respectively. The results clearly show that repellent activity
was dose dependent.
The results of the adulticidal activity of leaf methanol
extract of A. nilagiriga against the adult of two important
vector mosquitoes, viz., A. stephensi and A. aegypti are
presented in Table 4(Fig. 3). Among two vectors tested,
Table 1 Larval and pupal toxicity effect of methanol leaf extract of A. nilagirica against malarial vector, A. stephensi
Mosquito life stages Percentage of larval and pupal mortality ± SD LC
50
(LC
90
) 95 % confidence limit χ
2
(df04)
Concentration of A. nilagirica leaf extract (ppm) LC
50
LC
90
LFL-UFL UFL-UFL
200 300 400 500 600
First instar 41± 1.41 53 ± 1.01 69 ± 1.72 78 ± 1.85 94 ± 1.32 272.50 (590.07) 228.57–305.86 541.20–662.63 2.79*
Second instar 36± 1.01 48 ± 1.72 62 ± 1.41 72 ± 1.93 85 ± 1.85 311.40(688.81) 265.96–346.93 620.68–797.57 0.35*
Third instar 32± 1.72 42 ± 1.41 55 ± 1.16 65 ± 2.0 77± 0.74 361.51 (789.34) 318.06–399.48 697.83–945.34 0.12*
Fourth instar 26± 1.35 34 ± 1.16 45 ± 0.89 54 ± 1.41 69 ± 1.49 442.51 (901.59) 401.94–490.25 783.404–1,113.08 0.49*
Pupa 23± 1.41 32 ± 0.89 42 ± 1.01 52 ± 1.16 63 ± 0.63 477.23 (959.30) 433.80–535.21 824.19–1,209.86 0.01*
Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, χ
2
chi-square value, df degrees of freedom
*P<0.05 level, mean values of five replicates
Parasitol Res
the highest adulticidal activity was observed in highest
mortality followed by A. stephensi and A. aegypti. At higher
concentrations, the adult showed restless movement for
some times with abnormal wagging and died. The rates of
mortality were directly proportional to concentration. The
LC
50
and LC
90
values of A. nilagiriga leaf extracts against
adulticidal activity of A. stephensi and A. aegypti were the
following: A. stephensi LC
50
values were 205.78 ppm, and
A. aegypti LC
50
values were 242.52 ppm, respectively.
Discussion
Komalamisra et al. (2005) have reported that the petroleum
ether and methanol (MeOH) extracts of Rhinacanthus nasu-
tus and Derris elliptica exhibited larvicidal effects against A.
aegypti,C. quinquefasciatus,A. dirus, and Mansonia uni-
formis with LC
50
values between 3.9 and 11.5 mg/L, while
the MeOH extract gave LC
50
values of between 8.1 and
14.7 mg/L. D. elliptica petroleum ether extract showed LC
50
values of between 11.2 and 18.84 mg/L, and the MeOH
extract exhibited LC
50
values between 13.2 and 45.2 mg/L.
Earlier authors reported that the n-hexane, ethyl acetate, and
methanol extracts of Cassia nigricans showed 100 % larval
mortality against Ochlerotatus triseriatus (Georges et al.
2008). The mode of action of these leaf extract on mosquito
larvae are not known, but previous studies demonstrated that
phytochemicals interfered with the proper functioning of
mitochondria more specifically at the proton transferring
sites (Usta et al. 2002), and other studies by Rey et al.
(1999) and David et al. (2000) found that phytochemicals
primarily affect the midgut epithelium and secondarily af-
fect the gastric ceca and the malpighian tubules in mosquito
larvae. Furthermore, the crude extracts may be more effec-
tive compared to the individual active compounds due to
natural synergism that discourages the development of re-
sistance in the vectors (Maurya et al. 2007).
Sphaeranthus indicus,Cleistanthus collinus, and Murraya
koenigii leaf extracts were tested against the third-instar
larvae of Culex quinquefasciatus (Kovendan et al. 2012f).
Different parts of plants contain a complex of chemicals
with unique biological activity (Farnsworth and Bingel
1977), which is thought to be due to toxins and secondary
metabolites, which act as attractants or deterrents (Fisher
1991). Our result showed that leaf methanol extract of A.
nilagirica have significant larvicidal as well as repellent and
adulticidal activity. This results are comparable to earlier
reports of Jang et al. (2002) have reported that the methanol
extracts of Cassia obtusifolia,Cassia tora, and Vicia tetra-
sperma exhibited more than 90 % larval mortality at
0
10
20
30
40
50
60
70
80
90
100
% of larval and pupal mortality at 24 h
First instar Second instar Third instar Fourth instar Pupa First instar Second instar Third instar Fourth instar Pupa
A. stephensi A. aegypti
200
300
400
500
600
Fig. 2 Larval and pupal toxicity effect of leaf extract of A. nilagirica against A. stephensi and A. aegypti
Table 2 Larval and pupal toxicity effect of methanol leaf extract of A. nilagirica against dengue vector, A. aegypti
Mosquito life stages Percentage of larval and pupal mortality ± SD LC
50
(LC
90
) 95 % confidence limit χ
2
(df04)
Concentration of A. nilagirica leaf extract (ppm) LC
50
LC
90
LFL-UFL UFL-UFL
200 300 400 500 600
First instar 38 ± 1.32 47 ± 1.85 65±1.72 74±1.16 89±1.35 300.84 (646.67) 257.97–334.40 588.35–736.16 1.62*
Second instar 35 ± 1.41 41 ± 1.32 59±0.89 69±1.72 82±1.16 338.79 (726.07) 296.77–373.78 651.33–846.87 1.187
Third instar 28 ± 1.35 36 ± 1.85 53±1.41 62±0.74 74±1.01 394.69 (805.49) 356.52–432.18 714.52–957.50 0.52*
Fourth instar 21 ± 1.41 29 ± 1.20 42±1.01 54±1.16 65±1.72 470.74 (892.01) 432.59–518.77 783.28.1,078.33 0.10*
Pupa 17 ± 1.16 23 ± 0.89 34±1.41 48±1.01 55±0.74 542.11 (991.29) 495.21–613.03 855.40–1,237.08 0.55*
Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, χ
2
Chi-square value, df degrees of freedom
*P<0.05 level, mean values of five replicates
Parasitol Res
200 ppm on A. aegypti and Culex pipiens. The ethanolic
extract of whole plant L. aspera against the first to fourth instar
larvae and pupae values of LC
50
of I instar was 9.695 %, that of
II instarwas 10.272 %, that of III instar was 10.823 %, and that
of IV instar was 11.303 %, and pupae was 12.732 %, respec-
tively against A. stephensi (Kovendan et al. 2012a). The larvi-
cidal and adulticidal activities of ethanolic and water mixture
(50:50) of plant extracts Eucalyptus globulus,Cymbopogan
citratus,Artemisia annua,Justicia gendarussa,Myristica fra-
grans,Annona squamosa,andCentella asiatica were tested
against A. stephensi, and the most effective between 80 and
100 % was observed in all extracts (Senthilkumar et al. 2009).
In the present results, A. stephensi had the LC
50
and LC
90
values first to fourth instars larvae and pupae of 272.50,
311.40, 361.51, 442.51, and 477.23 ppm, and the LC
90
of
590.07, 688.81, 789.34, 901.59, and 959.30 ppm, respectively.
The methanol extract of Clerodendron inerme and
Acanthus ilicifolius at different concentrations (20–100 ppm)
and the LC
50
value of I–IV instars larvae and pupae were
45.74, 51.04, 57.17, 68.16, and 56.44 %, respectively; A.
ilicifolius leaf extract, LC
50
values of 69.579, 76.635,
82.692, 88.230, and 87.287 %, respectively (Kovendan and
Murugan 2011). Mathivanan et al. (2010) determine that the
LC
50
and LC
90
values of crude methanol extract of leaves of
Ervatamia coronaria on C. quinquefasciatus,A. aegypti,and
A. stephensi larvae in 24 h were 72.41, 65.67, and 62.08 and
136.55, 127.24, and 120.86 mg/L, respectively. Sakthivadivel
and Daniel (2008) showed the crude petroleum ether leaf
extract of Jatropha curcas to have larvicidal activity with
the LC
50
of <100 ppm on the early fourth instar larvae of
vector mosquitoes including C. quinquefasciatus,A. ste-
phensi,andA. aegypti. The leaf extract of A. alnifolia with
different solvents—hexane, chloroform, ethyl acetate, acetone
and methanol—were tested for larvicidal activity of against
mosquito vectors. The early fourth instar larvae of A. stephensi
had values of LC
50
0197.37, 178.75, 164.34, 149.90, and
Table 3 Repellent activity of methanol leaf extract of A. nilagirica against A. stephensi and A. aegypti
Mosquito species Concentration (ppm) Percentage of repellency±SD
Time post application of repellent (min)
30 60 90 120 150 180
A. stephensi 50 95.3± 1.4 87.2 ± 2.0 80.4 ± 1.4 76.4 ± 1.2 69.6± 2.1 61.1 ± 1.9
150 98.6± 1.1 90.2 ± 0.8 85.3 ± 2.0 83.1 ±1.0 74.3 ± 1.3 64.2 ± 0.4
250 100± 0.0 97.4± 0.9 93.6 ± 0.7 91.6 ± 0.8 85.4±0.4 73.4±1.4
350 100± 0.0 100± 0.0 98.6±0.0 95.6 ± 1.9 90.4±1.7 76.5±1.0
450 100± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 94.8± 1.2 91.2 ± 1.8
A. aegypti 50 91.6 ± 1.0 85.4 ± 1.3 77.6 ± 0.3 74.2 ± 1.4 69.1± 1.4 58.5 ± 1.9
150 96.2± 0.8 91.2 ± 1.9 82.1 ± 1.6 76.6 ± 1.9 67± 1.5 61.1 ±0.4
250 99.2± 0.9 98.6 ± 0.0 90.2 ± 1.9 87.9 ± 0.8 83.1± 1.9 70.6 ± 2.0
350 100± 0.0 100± 0.0 96.2±2.0 92.1 ± 1.3 82.5±0.4 73.6±0.1
450 100± 0.0 100 ± 0.0 100 ± 0.0 99.3 ± 0.4 90.2±0.5 86.2± 1.1
Table 4 Adulticidal activity of
methanol leaf extract of A. nila-
girica against mosquito vectors
LFL lower fiducidal limits, UFL
upper fiducidal limits, χ
2
chi-
square value
*P<0.05 level
Mosquito species Concentration
(ppm)
Mortality (%)
(mean± SD)
LC
50,
ppm
(LFL-UFL)
LC
90 ,
ppm
(LFL-UL)
χ
2
A. stephensi 100 27.2± 1.4 205.78 (175.
60–231.27)
459.51 (421.
68–511.44)
1.59*
200 53.6± 0.9
300 65.8± 2.7
400 84.6± 1.9
500 92.6± 2.1
Control 0.0± 0.0
A. aegypti 100 23.4±2.7 242.52 (212.
80–268.87)
523.73 (477.
50–589.32)
1.44*
200 46.8± 1.7
300 59.8± 2.9
400 74.2± 0.9
500 88.7± 1.4
Control 0.0± 0.0
Parasitol Res
125.73 ppm and LC
90
0477.60, 459.21, 435.07, 416.20, and
395.50 ppm, respectively. The A. aegypti hadvaluesofLC
50
0
202.15, 182.58, 160.35, 146.07, and 128.55 ppm and LC
90
0
476.57, 460.83, 440.78, 415.38, and 381.67 ppm, respective-
ly. The C. quinquefasciatus had values of LC
50
0198.79,
172.48, 151.06, 140.69, and 127.98 ppm and LC
90
0458.73,
430.66, 418.78, 408.83, and 386.26 ppm, respectively
(Kovendan et al. 2012b). Our results revealed that the larvi-
cidal effect of A. nilagiriga on A. aegypti had the LC
50
and
LC
90
values first to fourth instars larvae and pupae of 300.84,
338.79, 394.69, and 470.74 ppm, and the LC
90
646.67,
726.07, 805.49, 892.01, and 991.29 ppm, respectively.
According to our literature survey, there is no published
paper available on the repellent activity of A. nilagirica on
insects. However, in the same plant genus, benzene and
methanol extracts of Artemisia vulgaris have been reported
to have repellent activity against A. aegypti (Yit et al. 1985).
Amer and Mehlhorn (2006b) evaluated 41 plant extracts and
11 oil mixtures against the A. aegypti,A. stephensi, and C.
quinquefasciatus using the skin of human volunteers to find
out the protection time and repellency. The five most effec-
tive oils were those of Litsea (Litsea cubeba), Cajeput
(Melaleuca leucadendron), Niaouli (Melaleuca quinquener-
via), Violet (Viola odorata), and Catnip (Nepeta cataria),
which induced a protection time of 8 h at the maximum and
a 100 % repellency against all three species. Venkatachalam
and Jebanesan (2001) have also reported that the repellent
activity of methanol extract of Ferronia elephantum leaves
against A. aegypti activity at 1.0 and 2.5 mg/cm
2
concen-
trations gave 100 % protection up to 2.14 ± 0.16 h and 4.00±
0.24 h, respectively, and the total percentage protection was
45.8 % at 1.0 mg/cm
2
and 59.0 % at 2.5 mg/cm
2
for 10 h.
The essential oil of Zingiber officinalis showed repellent
activity at 4.0 mg/cm
2
, which provided 100 % protection
up to 120 min against C. quinquefasciatus (Pushpanathan et
al. 2008). Govindarajan (2010a) evaluate the larvicidal ac-
tivity of crude extract of Sida acuta against three important
mosquitoes with LC
50
values ranging between 38 and
48 mg/L; the crude extract had strong repellent action
against three species of mosquitoes, as it provided 100 %
protection against A. stephensi for 180 min followed by A.
aegypti (150 min). Our present investigations revealed that
leaf methanol extract of A. nilagirica have significant repel-
lent activity against A. stephensi and A. aegypti mosquitoes.
The highest concentrations of 450 ppm provided over 180
and 150 min protection in methanol extracts of A. nilagirica
and over 90 and 120 min protection in methanol extracts of
A. nilagirica against mosquito bites, respectively. Our
results showed that leaf extract of A. nilagirica have signif-
icant repellent activity against A. stephensi and A. aegypti
mosquitoes. The highest concentrations of 450 ppm provid-
ed over 180 and 150 min protection in methanol extracts of
A. nilagirica and over 90 and 120 min protection in meth-
anol extract of A. nilagirica against mosquito bites,
respectively.
A large number of synthetic chemical have been tested
for their repellent activity against mosquitoes. However, the
prohibitive retails cost of proprietary formulations of chem-
icals like N,N-diethyl-m-toluamide restricts their usage by
the poor in countries such as India. Hence, the search for a
safer, better, and cheaper repellent is an ongoing effort.
Since cost is an important factor, investigation on the use
of local plants as repellents is strongly recommended (Curtis
1990). Neem products are good mosquito repellents show-
ing 90–100 % protection against malaria vectors and about
70 % against C. quinquefasciatus (Sharma and Ansari
1994). One controlled study evaluated the efficacy of a
cream formulation containing 5 % neem oil against C.
quinguefasciatus and A. culicifacies. About 4–5gofthe
cream was applied to the exposed skin areas of human
volunteers in Ghaziabad, India in the summer months of
May/June and the monsoon months of August/September.
Neem cream was found to offer 82 % protection against
Culex bites and 100 % protection against Anopheles bites, as
compared to untreated controls (Nagpal et al. 2001). The
ethanolic extracts of the orange peel C. sinensis was tested
for the toxicity effect on the larvae of the yellow fever
mosquito A. aegypti (Amusan et al. 2005; Murugan et al.
2012).
Rajkumar and Jebanesan (2005) proved that the leaf
extract of C. asiatica has larvicidal properties and is an
inhibitor for adult emergence against C. quinquefasciatus.
The effects of the tested extract, adult emergence, and adul-
ticidal activity of the mosquitoes are remarkably greater
than those reported for other plant extracts in the literature.
For example at the highest concentration, 50 % inhibition of
the emergence of the adult mosquitoes was observed by the
use of the ethyl acetate fractions of Calophyllum inophyllum
seed and leaf, Solanum suratense and Samadera indica leaf
extracts, and the petrol ether fraction of Rhinocanthus
205.78
459.51
242.52
523.73
0
100
200
300
400
500
600
Concentration of plant extract (ppm
A. stephensi A. aegypti
LC50 LC90
Fig. 3 Adulticidal activity of leaf extract of A. nilagirica against A.
stephensi and A. aegypti expressed as LC
50
and LC
90
Parasitol Res
nasutus leaf extract on C. quinquefasciatus,A. stephensi,
and A. aegypti (Muthukrishnan et al. 1997). The above
findings support the results observed in this study the plant
A. nilagirica act as potent larvicide, adulticide as well as
disrupting the growth of larvae of A. stephensi and A.
aegypti. The LC
50
and LC
90
values of C. tora leaf extracts
against adulticidal activity of (hexane, chloroform benzene,
acetone, and methanol) C. quinquefasciatus,A. aegypti, and
A. stephensi were the following: for C. quinquefasciatus,
LC
50
values were 338.81, 315.73, 296.13, 279.23, and
261.03 ppm and LC
90
values were 575.77, 539.31, 513.99,
497.06, and 476.03 ppm; for A. aegypti,LC
50
values were
329.82, 307.3, and 252.03 ppm and LC
90
values were
563.24, 528.33, 36 496.92, 477.61, and 448.05 ppm; and
for A. stephensi,LC
50
values were 317.28, 300.30, 277.51,
263.35, and 251.43 ppm and LC
90
values were 538.22,
512.90, 483.78, 461.08, and 430.70 ppm, respectively
(Amerasan et al. 2012). Nathan et al. (2005) considered pure
limonoids of neem seed, testing for biological, larvicidal,
pupicidal, adulticidal, and antiovipositional activity, A. ste-
phensi, and the larval mortality was dose dependent with the
highest dose of 1 ppm azadirachtin evoking almost 100 %
mortality, affecting pupicidal and adulticidal activity and
significantly decreasing fecundity and longevity of A. ste-
phensi. The adult mortality was found in ethanol extract of
C. sinensis with the LC
50
and LC
90
values of 272.19 and
457.14 ppm, A. stephensi of 289.62 and 494.88 ppm, and A.
aegypti of 320.38 and 524.57 ppm, respectively (Murugan
et al. 2012). In the present adulticidal results, the LC
50
and
LC
90
values were 205.78 and 459.51 ppm for A. stephensi
and 242.52 and 523.73 ppm for A. aegypti, respectively.
In conclusion, the present study clearly proved that the leaf
extract of A. nilagirica has remarkable larvicidal, pupicidal as
well as repellent and adulticidal properties against A. stephensi
and A. aegypti vector mosquitoes. The flora of India has rich
aromatic plant diversity with potential for development of
natural insecticides for control of mosquito and other pests.
These results could encourage the search for new active natural
compounds offering an alternative to synthetic repellents and
insecticides from other medicinal plants. This leaf extracts of
A. nilagirica have the potential to be used as an ideal eco-
friendly approach for the vector control programmes.
Acknowledgments The authors are grateful to Mr. N. Muthukrishnan,
Technician, and Mr. A. Anbarasan, Lab Assistant, National Centre
for Diseases Control (NCDC), Mettupalayam, Tamil Nadu, for
their helping mosquito collection and mosquito samples provided
for the present work.
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