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Larvicidal, pupicidal, repellent and adulticidal activity of Citrus sinensis

orange peel extract against Anopheles stephensi, Aedesaegypti and Culex
quinquefasciatus (Diptera: Culi...

Article in Parasitology Research · July 2012

DOI: 10.1007/s00436-012-3021-8

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Parasitol Res
DOI 10.1007/s00436-012-3021-8
ORIGINAL PAPER
Larvicidal, pupicidal, repellent and adulticidal activity of Citrus
sinensis orange peel extract against Anopheles stephensi, Aedes
aegypti and Culex quinquefasciatus (Diptera: Culicidae)
Kadarkarai Murugan & Palanisamy Mahesh Kumar &
Kalimuthu Kovendan & Duraisamy Amerasan &
Jayapal Subrmaniam & Jiang-Shiou Hwang
Received: 26 May 2012 / Accepted: 19 June 2012
# Springer-Verlag 2012
Abstract Mosquitoes are the carriers of severe and well-
known illnesses such as malaria, arboviral encephalitis,
dengue fever, chikunguniya fever, West Nile virus and yel-
low fever. These diseases produce significant morbidity and
mortality in humans and livestock around the world. The
present study explored the effects of orange peel ethanol
extract of Citrus sinensis on larvicidal, pupicidal, repellent
and adulticidal activity against Anopheles stephensi, Aedes
aegypti and Culex quinquefasciatus. The orange peel mate-
rial was shade dried at room temperature and powdered
coarsely. From orange peel, 300 g powdered was macerated
with 1 L of ethanol sequentially for a period of 72 h each
and filtered. The yields of the orange peel ethanol crude
extract of C. sinensis 13.86 g, respectively. The extracts
were concentrated at reduced temperature on a rotary vacu-
um evaporator and stored at a temperature of 4 °C. The
larvicidal, pupicidal and adult mortality was observed after
24 h of exposure; no mortality was observed in the control
group. For C. sinensis, the median lethal concentration
values (LC50) observed for the larvicidal and pupicidal
activities against mosquito vector species A. stephensi first
to fourth larval instars and pupae were 182.24, 227.93,
291.69, 398.00 and 490.84 ppm; A. aegypti values were
92.27, 106.60, 204.87, 264.26, 342.45, 436.93 and
K. Murugan : P. Mahesh Kumar : K. Kovendan (*) :
D. Amerasan : J. Subrmaniam
Division of Entomology, Department of Zoology, School of Life
Sciences, Bharathiar University,
Coimbatore 641 046 Tamil Nadu, India
e-mail: gokulloyo@yahoo.co.in
J.-S. Hwang
Institute of Marine Biology, National Taiwan Ocean University,
Keelung 202-24, Taiwan
497.41 ppm; and C. quinquefasciatus values were 244.70,
324.04, 385.32, 452.78 and 530.97 ppm, respectively. The
results of maximum repellent activity were observed at
450 ppm in ethanol extracts of C. sinensis and the mean
complete protection time ranged from 150 to 180 min was
tested. The ethanol extract of C. sinensis showed 100 %
repellency in 150 min and showed complete protection in
90 min at 350 ppm against A. stephensi, A. aegypti and C.
quinquefasciatus, respectively. The adult mortality was
found in ethanol extract of C. sinensis with the LC50 and
LC90 values of 272.19 and 457.14 ppm, A. stephensi;
289.62 and 494.88 ppm, A. aegypti; and 320.38 and
524.57 ppm, respectively. These results suggest that the
orange peel extracts of C. sinensis have the potential to be
used as an ideal eco-friendly approach for the control of the
vector control programmes.
Introduction
Insect-transmitted disease remains a major source of illness
and death worldwide. Diseases that are healthcare-
associated transmission of viruses to human from mosqui-
toes are an expanding problem in tropical and subtropical
regions. Some of them such as dengue, malaria and West
Nile virus (WNV) are now the most frequent arboviral
diseases in the world (Wilder-Smith et al. 2009). Female
mosquitoes are one of the most world-wide important insect
pests that affect the health of human being and domestic
animals. Anopheles stephensi and Anopheles culicifacies are
the important vectors of malaria while Aedes aegypti and
Culex quinquefasciatus are the vectors of dengue and fila-
riasis, respectively. All these mosquito species have been
identified as primary vectors of the above diseases in this

arid region (Bansal et al. 1994). They require a blood meal
for egg production and produce a painful bite as they feed;
while feeding, they can transmit a number of diseases-
causing organisms to human and animals. These diseases
includes: encephalitis, dengue fever, filariasis, yellow fever
and malaria (Gouge et al. 2001).
Malaria remains one of the most prevalent diseases in the
tropical world. With 200 million to 450 million infections
annually worldwide, it causes up to 2.7 million deaths
(WHO 2010). In India, malaria is transmitted by six vector
species, in which A. stephensi is responsible in urban areas.
It is endemic in all parts of India, and periodic epidemics of
malaria occur every 5 to 7 years (Sharma 1996). Malaria
alone kills 3 million each year, including 1 child every 30 s
(Shell 1997). Malaria continues to be a major public health
problem in the tropical world. Of the total world population
of about 5.4 billion people, 2,200 million are exposed to
malarial infections in some 90 countries or areas. The most
recent estimates indicate that there may be 300–500 million
clinical cases each year, with countries in tropical Africa
accounting more than 90 % of these. Malaria is also the
cause of an estimated 1–4 to 2–6 million deaths worldwide
every year, with more than 90 % in Africa alone (WHO
1995; Snow et al. 1999; Breman 2001).
Dengue fever has become an important public health
problem as the number of reported cases continues to in-
crease, especially with more severe forms of the disease,
dengue hemorrhagic fever and dengue shock syndrome, or
with unusual manifestations such as central nervous system
involvement (Pancharoen et al. 2002). The yellow fever
mosquitoes, A. aegypti, are responsible for dengue fever in
India where the number of dengue fever cases has increased
significantly in recent years. Dengue viruses occur as four
antigenically related but distinct serotypes, which cause a
broad range of disease, including clinically asymptomatic
forms, classic dengue fever and the more severe forms such
as dengue hemorrhagic fever–dengue shock syndrome
(Fundacaõ Nacional de Saú de 2002). Agarwal et al. (2006)
observed a 4-year-old female child from northeastern India
with dengue encephalitis. To our knowledge, this is the first
reported case of dengue encephalitis from this region.
Lymphatic filariasis is a mosquito-borne disease caused
by mosquito-transmitted filarial nematodes, including
Wuchereria bancrofti and Brugia malayi. The infected peo-
ple carry the nocturnally periodic W. bancrofti, which has C.
quinquefasciatus as the main mosquito vector. C. quinque-
fasciatus is a vector of lymphatic filariasis, which is a
widely distributed tropical disease with around 120 million
people infected worldwide, and 44 million people have
common chronic manifestation (Bernhard et al. 2003). Cu-
lex is a genus of mosquito and is important in that several
species serve as vectors of important diseases, such as West
Nile virus, filariasis, Japanese encephalitis, St. Louis
Parasitol Res
encephalitis and avian malaria. The adult mosquito can
measure from 4 to 10 mm (0.16–0.4 in.), and morphologi-
cally has the three body parts common to insects: head,
thorax and abdomen. As a fly, it has one pair of wings. C.
quinquefasciatus breeds locally in storm sewer catch basins,
clean and polluted ground pools, ditches, animal waste
lagoons, effluent from sewage treatment plants and other
sites with organic wastes. C. quinquefasciatus is an oppor-
tunistic feeder and principal mosquito vector of WNV in this
metropolitan area (Goudarz Molaei 2007). According to
WHO, about 90 million people worldwide are infected with
W. bancrofti, the lymphatic dwelling parasite, and ten times
more people ate at the risk of being infected. In India alone, 25
million people harbor microfilaria and 19 million people
suffer from filarial disease manifestations (NICD 1990;
Kovendan et al. 2009).
Chemical control using synthetic insecticides had been
favourable so far because of their speedy action and easy
application. Certain plant species containing essential oils
have proved efficacy as larvicides, adulticides, ovicides and
repellents against different species of mosquitoes (Hwang et
al. 1985; Sharma et al. 1993; Mansour et al. 1998). Natural
pesticides, especially those derived from plants, are more
promising in this aspect. Aromatic plants and their essential
oils are very important sources of many compounds that are
used in different respects. Most of the mosquito control
programmes target the larval stage in their breeding sites
with larvicides (Knio et al. 2008). Personal protective meas-
ures, including repellents, are widely used to prevent the
transmission of mosquito-borne diseases by minimizing the
contact between humans and vectors (Pitasawat et al. 2003).
Repellent properties of several essential oil appear to be
associated with the presence of monoterpenoids and sesqui-
terpenes (Sukumar et al. 1991; Jaenson et al. 2006). Mono-
terpenes such as a-pinene, limonene, terpinolene, citronellol,
citronellal, camphor and thymol are common constituents of
a number of essential oil described in the literature as
presenting mosquito repellent activity (Ibrahim and Zaki
1998; Yang et al. 2004; Park et al. 2005).
The genus Citrus of the family Rutaceae includes
about 17 species distributed throughout the tropical and
temperate regions (Shaw 1977; Davies and Albrigo
1994). Citrus oils are mixtures of very volatile compo-
nents as terpenes and oxygenated compounds (Sato et al.
1996). Limonene, a monoterpene, is the major compo-
nent of lime and other related citrus essential oils (Lanças
and Cavicchioli 1990). Citrus sinensis (orange) is a small
flowering tree growing to about 10 m tall with evergreen
leaves, which are arranged alternately, of ovate shape with
crenulate margins and 4–10 cm long. The orange fruit is a
hesperidium, a type of berry. Like all citrus fruits, the
orange is acidic, with a pH level of around 2.5–3,
depending on the age, size and variety of the fruit.
Parasitol Res
Although this is not, on average, as strong as the lemon,
it is still quite strong on the scale—as strong as vinegar.
Orange oil is an essential oil produced by glands inside
the rind of an orange fruit. It is extracted or steam
distilled as a by-product of orange juice production. It is
composed of mostly (greater than 90 %) D-limonene
(Bauer et al. 2001) and is therefore often used in place
of pure D-limonene, which can be further extracted from
the oil by distillation. Many researchers have reported on
the effectiveness of plant extract against mosquito larvae
(Kalyanasundaram and Das 1985; Govindarajan et al.
2008; Kovendan and Murugan 2011; Kovendan et al. 2011;
2012c, d, e, g).
Taxonomy
Kingdom: Plantae
Phylum: Tracheophyta
Subphylum: Euphyllophytina
Class: Magnoliopsida
Subclass: Rosidae
Superorder: Rutanae
Order: Sapindales
Family: Rutaceae
Subfamily: Aurantioideae
Tribe: Aurantieae
Subtribe: Citrinae
Genus: Citrus
Species: sinensis
Botanical name: Citrus sinensis (Zipcode zoo 2012)
Essential oils are vegetable products whose constituents
are basically complex mixture of terpenic hydrocarbons and
oxygenated derivatives such as aldehydes, alcohols and
esters. These are accumulated mainly in secretary cavities
scattered throughout the fruit peels and leaf. Among many
sources, citrus fruit peels are the most familiar and rich
source of essential oils. Peels of citrus fruit comprise of
two layers, red outer layer as flavedo and inner white layer
as albedo (Nagi et al. 1977). The flavedo layer contains
essential oils in the range of 0.5 to 3.0 kg/ton of fruit (Sattar
and Mahmud 1992). Some of the Citrus species have been
reported as a source of botanical insecticides: as a variety of
these plants contain secondary metabolites that show insec-
ticidal activity against several coleopteran and dipteran (Su
et al. 1972; Sheppard 1984; Salvatore et al. 2004; Shrivas-
tava et al. 2010).
The present investigation was to explore the mosquito
control agent under laboratory conditions. The orange peels
extract C. sinensis for the control of malarial vector, A.
stephensi, dengue vector, A. aegypti, and filarial vector, C.
quinquefasciatus, was evaluated mosquito species.
Materials and methods
Collection of eggs and maintenance of larvae
The eggs of A. stephensi, A. aegypti and C. quinquefas-
ciatus were collected from 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 feed-
ing for 5 days. After blood feeding, enamel trays with
water from the culture trays were placed in the cage as
oviposition substrates.
Collection of plant and preparation of extract
The fresh orange fruit brought from Coonoor, Nilgiris dis-
trict, Tamil Nadu, were peeled and shopped with knife and
the soaked in distilled water. The fresh orange peels of C.
sinensis were shade dried at room temperature (28±2 °C)
for 10 to 15 days. The ground materials were obtained by
grinding the dry peels into a fine powdered separately using
commercial electrical blender. From orange peel, 300 g
powdered was macerated with 1 L of ethanol sequentially
for a period of 72 h each and filtered. The yields of the
orange peel ethanol crude extract of C. sinensis 13.86 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 100, 200, 300, 400 and
500 ppm, respectively.
 Parasitol Res

Larval/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
introduced into 500-mL glass beaker containing
249 mL of dechlorinated water and 1 mL of desired
concentrations of orange peel 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 by using Abbott’s
formula (Abbott’s 1925).

Observed mortality in treatment  Observed mortality in control

Corrected mortality ¼  100
100  Control mortality

Number of dead larvae=pupae

Percentage mortality ¼  100
Number of larvae=pupae introduced

The LC50 and LC90 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 and 450 ppm. For repellent
experiment, 50 laboratory-reared blood-starved adult female
mosquitoes that were between 3 and 10 days old were
placed into separate laboratory cages (45×45×40 cm). Be-
fore 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 application. The
different plant extracts being tested were applied from the
elbow to the fingertips. The arm was left undisturbed. An
arm treated with acetone and polysorbate 80 served as
control. The control and treated arms were introduced si-
multaneously into the cage. The numbers of bites were
counted over 5 min, every 30 min, from 1800 hours 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 considered a bite. If 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 volunteer
was used to nullify any effect of skin differences on repel-
lency. It was observed that there was no skin irritation from
the plant extract. The percentage protection was calculated
by using the following formula (Fradin and Day 2002;
Venkatachalam and Jebanesan 2001a, b).

ðfNo: of bites received by control armg  fNo: of bites received by treated armgÞ
Protection ¼  100
ðNo: of bites received by control armÞ

Adulticidal bioassay fresh prior to testing. The bioassay was conducted in an

Sugar-fed adult female mosquitoes (5 to 6 days old) were
used. The orange peel extract were diluted with acetone 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
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 mosqui-
toes before and after the exposure periods. 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 mosquitoes (20) were
Parasitol Res

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 24 h. Mor-
tality 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 larval, pupal and adult mortality data were
subjected to probit analysis for calculating LC50, LC90 and
other statistics at 95 % fiducial limits of upper fiducidal limit
and lower fiducidal limit, and Chi-square values were cal-
culated using the SPSS Statistical software package 16.0
version was used. Results with P<0.05 were considered to
be statistically significant.
Results
Larvicidal and pupicidal activity of orange peel ethanol
extract of C. sinensis at various concentrations against the
malarial vector, A. stephensi is given in the Table 1. Con-
siderable mortality was evident after the treatment of C.
sinensis for all larval instars and pupae. Mortality was
increased as the concentration increased, for example, in
first instars stage at 100 ppm concentration the larval mor-
tality was 39 %; whereas at 500 ppm concentration, it was
increased to 97 %. In pupal mortality at 100 ppm concen-
tration, it was 17 % increased to 52 % at 500 ppm (Fig. 1).
The LC50 and LC90 values were represented as follows:
LC50 value of first instar was 182.24 ppm, second instar
was 227.93 ppm, third instar was 291.69 ppm and fourth
instar was 398.00 ppm, respectively. LC90 value of first
instar was 452.44 ppm, second instar was 544.72 ppm, third
instar was 659.31 ppm and fourth instar was 858.92 ppm,
respectively. The LC50 value pupae was 490.84 ppm and
LC90 value pupae was 987.28 ppm, respectively.
Larval and pupal mortality of A. aegypti after the treatment
of orange peel ethanol extract of C. sinensis was observed.
Table 2 provides the larval and pupal mortality of A. aegypti
(first to fourth instars) after the treatment of A. aegypti at
different concentrations (100 to 500 ppm). Thirty-five percent
mortality was noted at first instars larvae by the treatment of C.
sinensis at 100 ppm, whereas it has been increased to 92 % at
500 ppm of C. sinensis extract treatment (Fig. 1). Similar trend
has been noted for all the instars of A. aegypti at different
concentration of C. sinensis treatment. The LC50 and LC90
values were represented as follows: LC50 value of first instar
was 204.87 ppm, second instar was 264.26 ppm, third instar
was 342.45 ppm and fourth instar was 436.93 ppm, respec-
tively. The LC90 value of first instar was 509.72 ppm, second
instar was 607.02 ppm, third instar was 734.98 ppm and
fourth instar was 891.63 ppm, respectively. The LC50 value
of pupae was 497.41 ppm, and the LC90 value of pupae was
938.06 ppm, respectively.
Larval and pupal mortality of C. quinquefasciatus after the
treatment of orange peel ethanol extract of C. sinensis was
observed. Table 3 shows the larval and pupal mortality of C.
quinquefasciatus (First to fourth instars and pupae) after the
treatment of C. quinquefasciatus at different concentrations
(100 to 500 ppm). Thirty-two percent mortality was noted at
first instars larvae by the treatment of C. sinensis at 100 ppm,
whereas it has been increased to 89 % at 500 ppm of C.
sinensis leaf extract treatment (Fig. 1). Similar trend has been
noted for all the instars of C. quinquefasciatus at different
concentration of C. sinensis treatment. The LC50 and LC90
values were represented as follows: LC50 value of first instar
was 244.70 ppm, second instar was 324.04 ppm, third instar

Table 1 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against malarial vector, A. stephensi

Mosquito % of Larval and pupal mortality±SD LC50 (LC90) 95 % confidence limit x2

life stages (df04)
Concentration of Citrus sinensis peel extract (ppm) LC50 LC90 UFL-UFL
(LFL-UFL)
100 200 300 400 500

First instar 39±1.41 52±1.85 66±1.01 83±1.32 97±1.16 182.24 (452.44) (147.11–210.44) (412.40–508.77) 4.52*
Second 32±1.32 46±1.41 58±1.72 74±1.85 89±1.15 227.93 (544.72) (192.86–257.57) (490.98–624.33) 1.35*
instar
Third instar 26±1.01 38±1.32 49±1.35 64±1.41 78±1.16 291.69 (659.31) (257.35–325.23) (583.27–779.67) 0.36*
Fourth 22±1.41 29±1.01 35±1.72 52±1.32 62±1.85 398.00 (858.92) (354.80–458.14) (728.24–1,095.55) 1.08*
instar
Pupa 17±1.01 22±1.16 29±1.85 41±1.41 52±1.60 490.84 (987.28) (432.00–591.37) (817.28–1,318.52) 0.41*

Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05, significant level; each value (mean±SD) five replicates
 Parasitol Res

Fig. 1 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against mosquito vectors

was 385.32 ppm and fourth instar was 452.78 ppm, respec-
tively. The LC90 value of first instar was 566.96 ppm, second
instar was 729.84 ppm, third instar was 806.57 ppm and
fourth instar was 890.08 ppm, respectively. The LC50 value
of pupae was 530.97 ppm, and the LC90 value of pupae was
967.19 ppm, respectively.
In the present observation, the results from the skin
repellent activity of orange peel ethanol extracts of C.
sinensis against blood starved adult female of A. stephensi,
A. aegypti and C. quinquefasciatus are given in Tables 4.
The present result shows that the percentage protection in
relation to dose and time (minutes). The highest concentra-
tions of 450 ppm provided over 180 and 150 min protection
in ethanol extracts of C. sinensis, and over 90 and 120 min
protection in ethanol extracts of C. sinensis against mosquito
bites, respectively. Lower concentrations provided 30 to
60 min of protection. The control provided only 3.2 ±
0.65 min of protection. However, we have observed repellen-
cy in ethanol extracts of C. sinensis, over 90 and 120 min
protection against A. stephensi, A. aegypti and C. quinquefas-
ciatus, respectively. In the present study, we observed 90 min
protection at 450 ppm in ethanol extract of C. sinensis against
A. stephensi, A. aegypti and C. quinquefasciatus. The results
clearly show that repellent activity was dose dependent.
The results of the adulticidal activity of ethanol extract of
C. sinensis against the adult of three important vector mosqui-
toes, viz., A. stephensi, A. aegypti and C. quinquefasciatus are
presented in Tables 5 (Fig. 2). Among three vectors tested, the
highest adulticidal activity was observed in high mortality
followed by A. stephensi, A. aegypti and C. quinquefasciatus.

Table 2 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against dengue vector, A. aegypti

Mosquito % of Larval and pupal mortality ± SD LC50 (LC90) 95 % confidence limit x2

life stages (df04)
Concentration of Citrus sinensis peel extract (ppm) LC50 LC90
(LFL-UFL) UFL-UFL
100 200 300 400 500

First 35±2.0 49±1.41 63±1.72 77±1.85 92±1.16 204.87 (509.72) (168.22–234.67) (460.97–580.90) 1.60*
instar
Second 29±1.01 40±1.85 52±1.67 69±1.32 83±1.41 264.26 (607.02) (230.24–295.22) (542.25–706.18) 0.09*
instar
Third 23±1.85 31±1.41 42±1.16 59±1.35 70±1.72 342.45 (734.98) (307.38–382.58) (642.38–887.13) 0.05*
instar
Fourth 19±1.72 26±1.54 31±1.41 43±1.87 61±1.16 436.93 (891.63) (390.09–507.51) (754.53.1,140.62) 1.94*
instar
Pupa 14±1.01 20±1.41 25±0.63 36±1.32 54±1.85 497.41 (938.06) (442.85–586.25) (791.90–1,205.04) 1.69*

Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 chi-square value, df degrees of freedom
*P<0.05, significant level; each value (mean±SD) five replicates
Parasitol Res

Table 3 Larval and pupal toxicity effect of orange peel ethanol extract of C. sinensis against filarial vector, C. quinquefasciatus

Mosquito % of Larval and pupal mortality ± SD LC50 (LC90) 95 % confidence limit x2

life stages (df04)
Concentration of Citrus sinensis peel extract (ppm) LC50 LC90
(LFL-UFL) UFL-UFL
100 200 300 400 500

First 32±1.16 41±1.41 56±1.35 69±1.93a 89±1.72 244.70 (566.96) (210.98–274.23) (509.94–652.10) 3.57*
instar
Second 26±1.41 35±1.16 43±1.01 58±1.85 74±1.32 324.04 (729.84) (287.75–363.51) (635.46–887.18) 1.37*
instar
Third 20±1.32 29±1.72 38±1.41 51±1.16 65±1.49 385.32 (806.57) (346.34–436.38) (694.83–998.69) .274*
instar
Fourth 16±0.48 22±1.85 33±1.41 42±1.01 57±1.72 452.78 (890.08) (405.48–524.86) (756.65–1,128.82) .353*
instar
Pupa 11±1.41 16±1.01 26±1.16 31±1.85 49±1.72 530.97 (967.19) (470.76–632.78) (813.45–1,251.91) 1.13*

Control nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05, significant level; each value (mean±SD) five replicates

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
LC50 and LC90 values were 272.19 and 457.14 ppm, A.
stephensi; 289.62 and 494.88 ppm, A. aegypti; 320.38 and
524.57ppm, respectively.
Discussion
Singhi et al. (2006) have reported the latex of C. procera has
shown larvicidal efficacy against all three important vector
species: A. aegypti, A. stephensi and C. quinquefasciatus in
India. Patil et al. (2011) evaluated larvicidal activity of
extracts of medicinal plants Plumbago zeylanica and Ces-
trum nocturnum against A. aegypti; the LC50 values of both
the plants were less than 50 ppm. The larvicidal stability of
the extracts at five constant temperatures (19 °C, 22 °C, 25 °C,
28 °C and 31 °C) evaluated against fourth instars larvae
revealed that toxicity of both plant extracts increases with
increase in temperature. Halim (2008) has reported the insec-
ticidal activity of Zingiber officinale against the larval matu-
ration, and adult emergency of Anopheles pharoensis third
stage was evaluated the concentrations of 100 %, 70 %, 50 %,

Table 4 Repellent activity of orange peel ethanol extract of C. sinensis against mosquito vectors

Mosquito species Concentration (ppm) % of repellency±SD

Time post application of repellent (min)

30 60 90 120 150 180

A. stephensi 50 99.4±0.8 89±1.0 83.8±1.6 74.6±1.7 67.2±1.4 58.4±1.3

150 100±0.0 98±1.6 89.8±1.4 79.2 ±1.1 72.8±1.7 63±1.4
250 100±0.0 99.6±0.4 96±1.6 87.6±1.8 80.2±1.1 71.8±1.4
350 100±0.0 100±0.0 100±0.0 91±1.4 86.6±1.8 77.6±1.0
450 100±0.0 100±0.0 100±0.0 100±0.0 92.4±1.8 88.6±1.2
A. aegypti 50 94.4±1.3 87.8±1.7 79±1.4 72.8±1.6 64.2±1.5 54±1.4
150 99±0.8 92.4±1.8 85.6±1.6 74.2±1.7 67±1.4 60.6±0.4
250 100±0.0 100±0.0 92.6±1.7 83.4±1.4 76.2±1.1 68.6±1.0
350 100±0.0 100±0.0 98.4±1.3 87.6±1.8 78.2±0.7 71.6±0.8
450 100±0.0 100±0.0 100±0.0 98.2±1.7 88.6±1.4 77.2±1.1
C. quinquefasciatus 50 91.2±1.1 83.8±1.4 76.4±1.0 64.6±1.8 56.2±1.6 49±0.6
150 97.2±1.9 90.6±1.8 82±1.4 72.2±1.5 62±1.4 56.6±2.0
250 100±0.0 94.8±1.7 88±1.2 82.2±1.1 72±1.3 63.6±1.3
350 100±0.0 100±0.0 91±0.8 85.4±1.8 76.6±1.3 67.8±1.7
450 100±0.0 100±0.0 97.6±1.0 91±0.8 82.4±1.3 73.2±1.5
 Parasitol Res

Table 5 Adulticidal activity of

orange peel ethanol extract of C. Mosquito Concentration Mortality (%) LC50, ppm LC90, ppm x2
sinensis against mosquito species (ppm) (Mean ± SD) (LFL-UFL) (LFL-UL)
vectors
A. stephensi 180 24.2±2.0 272.19 457.14 1.27*
260 50.4±2.7 (250.93–290.73) (429.68–494.00)
340 67.8±3.4
420 82.5±2.2
500 95.1±1.7
Control 0.0±0.0
A. aegypti 180 21.7±1.4 289.62 494.88 2.19*
260 47.8±2.3 (267.53–309.24) (462.58–539.43)
340 62.6±2.2
420 76.2±2.6
500 91.4±2.5
Control 0.0±0.0
C. quinquefasciatus 180 17.2±2.3 320.38 524.57 0.49*
260 37.4±2.1 (300.29–339.55) (490.31–571.84)
340 55.9±2.0
LFL lower fiducidal limits, UFL 420 72.4±1.4
upper fiducidal limits, x2 Chi- 500 86.8±2.9
square value
Control 0.0±0.0
*P<0.05, significant level

25 %, 5 %, 2 %, 1 %, 0.9 %, 0.7 %, 0.5 %and 0.3 % showed
100 % larval mortality rate and at 0.2 % and 0.1 % caused
mortality of 66.7 %, respectively. The ethanolic extract of
whole plant Leucas aspera against the first to fourth instar
larvae and pupae values of LC50 0I instar was 9.695 %, II
instar was 10.272 %, III instar was 10.823 % and IV instar was
11.303 %, and pupae was 12.732 %, respectively against A.
stephensi (Kovendan et al. 2012a). In the present results, A.
stephensi had the LC50 and LC90 values first to fourth instars
larvae and pupae of 182.24, 227.93, 291.69, 398.00 and
490.84 ppm, respectively, and the LC90 452.44, 544.72,
659.31, 858.92 and 987.28 ppm, respectively.
Yadav et al. (2002) have reported the methanol, chloroform
and ether extracts of Euphorbia tirucalli latex and stem bark
were evaluated for larvicidal activity against laboratory-reared
larvae of C. quinquefasciatus. Sharma et al. (2005) reported
that the acetone extract of Nerium indicum and Thuja
oriertelis has been studied with LC50 values of 200.87,
127.53, 209.00 and 155.97 ppm against III instar larvae of
A. stephensi and C. quinquefasciatus, respectively. Clitoria
ternatea leaf methanol extract showed dose-dependent larvi-
cidal activity against A. stephensi with LC50 values of 555.6
(24 h) and 867.3 (48 h)ppm, also the root extracts with LC50
value of 340 ppm (48 h). Seed extract showed larvicidal
activity (LC50 0116.8, 195 ppm) after 24 h and (LC50 065.2,
154.5 ppm) after 48 h treatment against A. stephensi and A.
aegypti, respectively. Larvicidal activity of flower methanol
extract showed LC50 values 233 and 302.5 ppm against A.
stephensi and A. aegypti, respectively, after 48 h treatment.
Methanol extract showed lowest LD values against several
instar of larvae and 50 adult (121.59, 142.73, 146.84, 202.98,
290.65, 358.42 and 300.03 μg/cm2, respectively) which indi-
cates highest toxicity or insecticidal activity (Ashraful Alam et
al. 2009). Sphaeranthus indicus LC50 values were 544.93,

Fig. 2 Adulticidal activity of

orange peel ethanol extract of
C. sinensis against mosquito
vectors
Parasitol Res
377.86 and 274.79 ppm and LC90 values were 1,325.32,
1,572.55 and 1,081.29 ppm at 24 h; Cleistanthus collinus
LC50 values were 375.34, 318.29 and 226.10 ppm, and LC90
values were 699.65, 1,577.62 and 1,024.92 ppm at 24 h; and
Murraya koenigii LC50 values were 963.53, 924.85 and
857.62 ppm and LC90 values were 1,665.12, 1,624.68 and
1,564.37 ppm at 24 h, respectively. However, the highest
larval mortality was observed in C. collinus followed by S.
indicus and M. koenigii of various concentrations at 24, 48 and
72 h against C. quinquefasciatus (Kovendan et al. 2012f). The
citrus leaf extracts from hexane possessed moderate larvicidal
efficiency respectively against dengue vector. The bioassays
resulted in an LC 50 and LC 90 value of 446.84 and
1370.96 ppm, respectively after 24 h of exposure. However,
the extracts were proved to be remarkable irritant against
adults A. aegypti, more pronounced effects being observed
on blood-fed females than unfed females (Radhika et al.
2012).). Kovendan et al. (2011) reported that Bacillus thur-
ingiensis against the first to fourth-instar larvae of values
LC50 09.332 %, 9.832 %, 10.212 % and 10.622 %, and
LC90 015.225, 15.508, 15.887 and 15.986 larvae of C. quin-
quefasciatus, respectively. LC50 value of I to IV instars and
pupae 155.29, 198.32, 271.12, 377.44 and 448.41 ppm re-
spectively. In the present results, A. aegypti had the LC50 and
LC90 values first to fourth instars larvae and pupae of 204.87,
264.26, 342.45, 436.93 and 497.41 ppm, respectively, and the
LC90 509.72, 607.02, 734.98, 891.63 and 938.06 ppm,
respectively.
The methanol extract of Clerodendron inerme and Acan-
thus ilicifolius at different concentrations (20 to 100 ppm)
and the LC50 value of I to IV instars larvae and pupae were
45.749 %, 51.04 %, 57.170 %, 68.166 % and 56.444 %,
respectively, A. ilicifolius leaf extract, LC50 values of
69.579 %, 76.635 %, 82.692 %, 88.230 % and 87.287 %,
respectively (Kovendan and Murugan 2011). Khanna et al.
(2011) have reported that the larvicidal crude leaf extract of
Gymnema sylvestre showed the highest mortality in the
concentration of 1,000 ppm against the larvae of Anopheles
subpictus (LC50 0166.28 ppm) and against the larvae of C.
quinquefasciatus (LC50 0186.55 ppm), and the maximum
efficacy was observed in gymnemagenol compound isolated
from petroleum ether leaf extract of G. sylvestre with LC50
values against the larvae of A. subpictus at 22.99 ppm and
against C. quinquefasciatus at 15.92 ppm, respectively. The
leaf extract of Acalypha alnifolia with different solvents—
hexane, chloroform, ethyl acetate, acetone and methanol
were tested for larvicidal activity of against mosquito vec-
tors. The early fourth instar larvae of A. stephensi had values
of LC50 0197.37, 178.75, 164.34, 149.90 and 125.73 ppm
and LC90 0477.60, 459.21, 435.07, 416.20 and 395.50 ppm,
respectively. The A. aegypti had values of LC50 0202.15,
182.58, 160.35, 146.07 and 128.55 ppm and LC90 0476.57,
460.83, 440.78, 415.38 and 381.67 ppm, respectively. The
C. quinquefasciatus had values of LC50 0198.79, 172.48,
151.06, 140.69 and 127.98 ppm and LC90 0458.73, 430.66,
418.78, 408.83 and 386.26 ppm, respectively (Kovendan et
al. 2012b). In the present results, C. quinquefasciatus had
the LC50 and LC90 values first to fourth instars larvae and
pupae of 244.70, 324.04, 385.32, 452.78 and 530.97 ppm,
respectively and the LC90 566.96, 729.84, 806.57, 890.08,
967.19 ppm, respectively.
Venkatachalam and Jebanesan (2001a, b) 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 concentrations 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 Tagetes
minuta, providing a repellency of 90 % protection for 2 h
against A. stephensi, C. quinquefasciatus and A. aegypti,
was reported by Tyagi et al. (1994). It was found that a
CO2 extract of the seeds of the Mediterranean plant Vitex
agnus castus can be used as a spray to keep away especially
Ixodes ricinus and Rhipicephalus sanguineus ticks from
animals and humans for at least 6 h. In addition, mosquitoes,
biting flies and fleas are also repelled for about 6 h
(Mehlhorn et al. 2005). Previously, Curcurbita maxima seeds
were shown to have diversified biological functions, such as
insecticidal properties against certain agricultural pests (Zhou
et al. 2000. Larvicidal, ovicidal, and repellent activities of the
leaf extract of C. maxima plants against C. quinquefasciatus
(Say) (Mullai and Jebanesan 2007) and also for organic form-
ing (Shrivastava et al. 2010) have been reported. C. sinensis
and Citrus aurantifolia were shown to contain insecticidal
activity against mosquito, cockroach and housefly (Ezeonu
et al. 2001). Kuppusamy and Ayyadurai (2012) reported that
lyophilized powders of purified Cyt1A crystals of B. thurin-
giensis were much more toxic yielding a 50 % LC50 of
11.332 mg/L, respectively.
There are few published studies using essential oils or
plant derivatives for repellency against mosquitoes. Many
different techniques have been used to measure mosquito
repellency. Besides different techniques, there are differen-
ces in biting pressure in a mosquito population which is
another factor affecting repellency testing (Barnard et al.
1998). Rosmarinus officinalis was effective in terms of
repellence time against A. aegypti in concentrations as low
as 12.5 %. These results are consistent with reports of insect
repellent properties of rosemary, including Culex pipiens
pallens (Choi et al. 2002). The mosquito sensitivity to
repellents varies among Aedes, Anopheles and Culex mos-
quitoes (Amer and Mehlhorn 2006a, b). A large number of
plant extracts have been reported to have mosquitocidal or
repellent activity against mosquito vectors, but very few
plant products have shown practical utility for mosquito
control. Some of the plants that have been tested against

mosquito larvae in India are Cleome viscosa, Ocimum basili-
cum, Vitex negundo, Delonixregia, Oligo chaetaramosa, Aza-
dirachta indica, Quassia amara, Anacardium occidentale,
Thevetianerii folia etc. Natural products are preferred because
of their biodegradability and less toxic compared to the syn-
thetic ones (Datta et al. 2010).
Benzene and methanol extracts of Artemisia vulgaris
have been reported to have repellent activity against A.
aegypti 17 (Yit et al. 1985). Quelling, the insect repellent
produced in China, derived from the extract of the lemon
grass and eucalyptus plants were evaluated against mosqui-
toes. Essential oil obtained from Vitex negundo was used as
repellent against A. aegypti (Hebbalkar et al. 1992). Neem
products are good mosquito repellents showing 90 % to
100 % protection against malaria vectors and about 70 %
against C. quinquefasciatus (Ansari and Razdan 1994). One
controlled study evaluated the efficacy of a cream formula-
tion containing 5 % neem oil against C. quinquefasciatus
and A. culicifacies. The cream (4–5 g) 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 % pro-
tection against Anopheles bites, as compared to untreated
controls (Nagpal et al. 2001). The ethanolic extracts of the
orange peel (C. sinensis) were tested for the toxicity effect
on the larvae of the yellow fever mosquito A. aegypti
(Amusan et al. 2005); susceptibility tests were carried out
in C. quinquefasciatus larvae using peel oil extracts of
Citrus aurantium, C. sinensis and Citrus limon (Mwaiko
1992); volatile extracts of C. sinensis had insecticidal activ-
ity against mosquito, cockroach and housefly (Ezeonu et al.
2001). Garcia and Desrochers (1979) observed appreciable
mortality only with high concentrations (1×107cells/ml) of
B. thuringiensis var. israelensis. The biocide at 1 to 10 kg/ha
(0.25 to 2.5 ppm) caused 18 % to 88 % mortality of midges
during a 4-week evaluation period. Mahesh Kumar et al.
(2012) have reported that the LC50 values of B. thuringien-
sis against the first to fourth instar larvae and pupae were
133.88, 157.14, 179.44, 206.80 and 240.74 ppm; and the
LC90 values were 321.04, 346.89, 388.86, 430.95 and
492.70 ppm, respectively
Ansari et al. (2000) reported that the peppermint oil gave
94.1 % protection for 6 h, while mylol oil gave 95 %
protection for 7.2 h. They also reported that mylol oil and
peppermint oil gave 100 % protection for 11 h against
Anopheles annularis. Mylol oil gave 95.4 % protection for
8.7 h for the Anopheline species, whereas the peppermint oil
gave 86.3 % protection for 8 h. Phukan and Kalita (2005)
showed that Litsea salicifolia recorded 70 % and 50 %
repellency for 3 and 4 h, respectively, against A. aegypti,
but they failed to show much activity against C. quinque-
fasciatus. Mullai et al. (2008) have also reported that the
Parasitol Res
skin repellent test at 1.0, 2.5 and 5.0 mg cm2 concentration
gave the mean complete protection time ranged from 119.17
to 387.83 min against A. stephensi with the benzene, petro-
leum ether, ethyl acetate and methanol extracts of Citrullus
vulgaris tested. Our results showed that orange peel extract
of C. sinensis have significant repellent activity against A.
stephensi, A. aegypti and C. quinquefasciatus mosquitoes.
The highest concentrations of 450 ppm provided over 180
and 150 min protection in ethanol extracts of C. sinensis;
over 90 and 120 min protection in ethanol extracts of C.
sinensis against mosquito bites, respectively.
The effects of the tested extract, adult emergence and
adulticidal 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 nasu-
tus leaf extract on C. quinquefasciatus, A. stephensi and A.
aegypti (Muthukrishnan and Puspalatha 2001). Similarly
88 % of the adult mortality was observed by the use of
Pelargonium citrosa leaf extracts at 2 % concentration
against A. stephensi (Jeyabalan et al. 2003). The adults that
emerged from all the treatments were deformed due to
inhibition and disruption of normal physiological and met-
abolic processes (Murugan et al. 1996). Similar result was
obtained in the root extract of Valeriana jatamansi which
exhibited adulticidal activity of 90 % lethal concentration
against adult A. stephensi, A. culicifacies, A. aegypti, A.
albopictus and C. quinquefasciatus and were 0.14, 0.16,
0.09, 0.08 and 0.17, and 0.24, 0.34, 0.25, 0.21 and
0.28 mg/cm2, respectively (Dua et al. 2008).
Nathan et al. (2005) considered pure limonoids of neem
seed, testing for biological, larvicidal, pupicidal, adulticidal
and antiovipositional activity, A. stephensi and the larval
mortality was dose-dependent with the highest dose of
1 ppm azadirachtin evoking almost 100 % mortality, affect-
ing pupicidal and adulticidal activity and significantly de-
creased fecundity and longevity of A. stephensi. The highest
adulticidal effect was established from Piper sarmentosum,
followed by Piper ribesoides and Piper longum, with LD50
values of 0.14, 0.15 and 0.26 μg/female, respectively
(Choochote et al. 2006). This result is also comparable to
earlier reports of Dua et al. (2010) who observed that the
adulticidal activity of the essential oil of Lantana camara
was evaluated against different mosquitoes species on
0.208 mg/cm2 impregnated papers, and the KDT50 and
KDT90 values of the essential oil were 20, 18, 15, 12 and
14 min, and 35, 28, 25, 18 and 23 min against A. aegypti, C.
quinquefasciatus, A. culicifacies, Anopheles fluvialitis and
A. stephensi with their percent mortality of 93.3 %, 95.2 %,
100 %, 100 % and 100 %, respectively. In the present
Parasitol Res
adulticidal results, the LC50 and LC90 values were 272.19
and 457.14 ppm, A. stephensi; 289.62 and 494.88 ppm, A.
aegypti; 320.38 and 524.57 ppm, respectively.
In conclusion, the present study clearly proved that the
efficacy of orange peel extracts of C. sinensis can be sug-
gested as a larvicidal, pupicidal, repellent and adulticidal
activity against A. aegypti, A. stephensi and C. quinquefas-
ciatus as target species. The results reported the efficacy for
controlling mosquitoes and mortality properties of natural
product extracts since they are considered as environmen-
tally safe and eco-friendly approaches for the vector control
programmes.
Acknowledgments We thank to Dr. K. Sasikala, Professor and Head,
Department of Zoology, Bharathiar University for the laboratory facil-
ities provided. 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 help-
ing mosquito collection and mosquito samples provided for the present
work.
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