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Parasitol Res (2012) 110:669678
DOI 10.1007/s00436-011-2540-z
ORIGINAL PAPER
Bioefficacy of larvicdial and pupicidal properties
of Carica papaya (Caricaceae) leaf extract and bacterial
insecticide, spinosad, against chikungunya vector, Aedes
aegypti (Diptera: Culicidae)
Kalimuthu Kovendan & Kadarkarai Murugan &
Arjunan Naresh Kumar & Savariar Vincent &
Jiang-Shiou Hwang
Received: 20 June 2011 / Accepted: 30 June 2011 / Published online: 13 July 2011
# Springer-Verlag 2011
Abstract The present study was carried out to establish the
properties of Carica papaya leaf extract and bacterial
insecticide, spinosad on larvicidal and pupicidal activity
against the chikungunya vector, Aedes aegypti. The
medicinal plants were collected from the area around
Bharathiar University, Coimbatore, India. C. papaya leaf
was washed with tap water and shade-dried at room
temperature. An electrical blender powdered the dried plant
materials (leaves). The powder (500 g) of the leaf was
extracted with 1.5 l of organic solvents of methanol for 8 h
using a Soxhlet apparatus and then filtered. The crude leaf
extracts were evaporated to dryness in a rotary vacuum
evaporator. The plant extract showed larvicidal and pupici-
dal effects after 24 h of exposure; however, the highest
larval and pupal mortality was found in the leaf extract of
methanol C. papaya against the first- to fourth-instar larvae
and pupae of values LC50 =I instar was 51.76 ppm, II instar
was 61.87 ppm, III instar was 74.07 ppm, and IV instar was
82.18 ppm, and pupae was 440.65 ppm, respectively, and
K. Kovendan (*) : K. Murugan : A. Naresh 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
S. Vincent
Tamil Nadu State Council for Science and Technology,
DOTE Campus, Guindy,
Chennai 600 025( Tamil Nadu, India
J.-S. Hwang
Institute of Marine Biology, National Taiwan Ocean University,
Keelung 202-24, Taiwan
bacterial insecticide, spinosad against the first to fourth
instar larvae and pupae of values LC50 =I instar was
51.76 ppm, II instar was 61.87 ppm, III instar was
74.07 ppm, and IV instar was 82.18 ppm, and pupae
was 93.44 ppm, respectively. Moreover, combined treat-
ment of values of LC50 =I instar was 55.77 ppm, II instar
was 65.77 ppm, III instar was 76.36 ppm, and IV instar
was 92.78 ppm, and pupae was 107.62 ppm, respectively.
No mortality was observed in the control. The results that
the leaves extract of C. papaya and bacterial insecticide,
Spinosad is promising as good larvicidal and pupicidal
properties of against chikungunya vector, A. aegypti. This
is an ideal eco-friendly approach for the control of
chikungunya vector, A. aegypti as target species of vector
control programs.
Introduction
Mosquitoes constitute a major public health problem as
vectors of serious human like malaria, filariasis, Japanese
encephalitis, dengue fever, chikunkunya, and yellow fever
cause substantial mortality and morbidity among people
living in tropical and subtropical zones (Jang et al. 2002).
The container-breeding mosquito, Aedes aegypti L. thrives
in urban and peridomestic environments where it transmits
the dengue virus to humans (Gubler 1998). A. aegypti L. is
generally known as a vector for an arbovirus responsible for
dengue and chikunkunya, which is endemic to South Asia,
the Pacific island area, Africa, and the Americas. This
mosquito is also a vector of yellow fever in Central and
South America and West Africa.
670
Dengue fever has become an important public health
problem as the number of reported cases continue to
increase, especially with more severe of the disease, dengue
hemorrhagic fever and dengue shock syndrome, or with
unusual manifestations such as central nervous system
involvement (Pancharoen et al. 2002). It is estimated
that there are between 50 and 100 million cases of
dengue fever (DF) and about 500,000 cases of dengue
hemorrhagic fever (DHF) each year which require
hospitalization (Maheswaran et al. 2008). Dengue fever
is spread through the bite of an infected A. aegypti
mosquito. The mosquito gets the virus by biting an
infected person. The first symptom of the disease appears
in about 57 days after the infected mosquito bites a
healthy person. It is possible to become infected by
dengue multiple times because the virus has four different
serotypes. The dengue symptoms of dengue fever include
high fever, rash, and a severe headache. Additional of
chickungunya fever symptoms include severe joint and
muscular pain (breakbone fever), nausea, vomiting, and
eye pain. Although dengue fever itself is rarely fatal, it
can be an extraordinary painful and disabling illness and
may become epidemic in a population following the
introduction of a new serotype (MorenaSanchez et al.
2006).
Carica papaya, belongs to the family of Caricaceae, and
several species of Caricaceae have been used as remedy
against a variety of diseases (Mello et al. 2008; Munoz et
al. 2000). Originally derived from the southern part of
Mexico, C. papaya is a perennial plant, and it is presently
distributed over the whole tropical area. In particular, C.
papaya fruit circulates widely, and it is accepted as food or
as a quasi drug. Many scientific investigations have been
conducted to evaluate the biological activities of various
parts of C. papaya, including fruits, shoots, leaves, rinds,
seeds, roots or latex. The leaves of papaya have been
shown to contain many active components that can
increase the total antioxidant power in blood and reduce
lipid peroxidation level, such as papain, chymopapain,
cystatin, -tocopherol, ascorbic acid, flavonoids, cyanogenic
glucosides, and glucosinolates (Seigler et al. 2002).
Fruit and seed extracts have pronounced bactericidal
activities (Emeruwa 1982). Leaves have been poulticed into
nervous pains, elephantoid growths, and it has been smoked
for asthma relief amongst tropical tribal communities. The
hypoglycemic effect of ethanolic extract of unripe, mature
fruits has been reported by Olagunju et al. (1995). Moreover,
C. papaya leaf juice is consumed for its purported anti-
cancer activity by people living on the Gold Coast of
Australia, with some anecdotes of successful cases being
reported in various publications. C. papaya leaf extracts have
also been used for a long time as an aboriginal remedy for
various disorders, including cancer and infectious diseases.
Parasitol Res (2012) 110:669678
C. papaya contains two important biologically active
compounds vis: chymopapain and papain which are
widely used for digestive disorders (Huet et al. 2006).
It showed that papaya-derived papain, caricain, chymopain,
and glycerin endopeptidase can improve acidic pH
conditions and pepsin degradation. Other active com-
pounds of C. papaya are lipase, or CPL, a hydrolase,
which is tightly bonded to the water-insoluble fraction of
crude papain and is thus considered as a naturally
immobilized biocatalyst (Dominguez et al. 2006).
According to the folk medicine, papaya latex can cure
dyspepsia and also applicable for external burns and
scalds. Seeds and fruits are excellent anthelminthic and
anti-amoebic (Okeniyi et al. 2007). Dried and pulverized
leaves are sold for making tea; also the leaf decoction is
administered as a purgative for horses and used for the
treatment of genetic-urinary system. Unripe and semi-ripe
papaya fruits are ingested or applied on the uterus to
cause absorption. However, the consumption of unripe
and semi-ripe papaya fruits could be unsafe during
pregnancy causes no risk (Adebowale et al. 2002).
The larvicidal properties of crude extracts of three plants,
viz. C. papaya, Murraya paniculata, and Cleistanthus
collinus against Culex quinquefasciatus as target species.
The relative efficacy of the plant extracts in vector control
was as follows: C. papaya seed extract>M. paniculata fruit
extract>M. paniculata leaf extract>C. collinus leaf extract.
To examine the potential role of C. papaya as anti-cancer
therapy, we analyzed in this report the anti-tumor activity
of the aqueous extract of the leaves of C. papaya (CP)
against various cancer cell lines, as well as its potential
immunomodulatory effects, and attempted to identify the
active components (Rawani et al. 2009).
In fact, many researchers have reported on the effectiveness
of plant extracts or essential oils against mosquito larvae
(Mehlhorn et al. 2005; Amer and Mehlhorn 2006a, b;
Rawani et al. 2009 a, b). Govindarajan (2009) reported
that the leaf methanol, benzene, and acetone extracts of
Cassia fistula were studied for the larvicidal, ovicidal, and
repellent activities against A. aegypti. Daniellia oliveri is
traditionally used to reduce numbers of mosquitoes
indoors at night (Curtis et al. 1991).
Spinosad is a natural fermentation product produced by
an actinomycete, Saccharopolyspora spinosa Mertz and
Yao. This compound is a mixture of spinosyns A and D. It
has shown activity against Lepidoptera, Thysanoptera, and
other insect orders such as Diptera. This naturally derived
insecticide has been reported to have no adverse effects on
predatory insects such as ladybirds, lacewings, big-eyed
bugs, or minute pirate bugs (Kirst et al. 1992; DeAmicis et al.
1997; Copping and Menn 2001; Williams et al. 2003).
Spinosad acts as a stomach and contact poison and degrades
rapidly in the environment (Cisneros et al. 2002). An
Parasitol Res (2012) 110:669678
immediate effect of ingestion is the cessation of feeding,
followed 24 h later, by paralysis and death. This
compound is a neurotoxin with a novel mode of action
involving the nicotinic acetylcholine receptor and GABA
receptors (Watson 2001). Spinosad has little toxicity to
birds and mammals (Bret et al. 1997; Breslin et al. 2000).
There is no cross-resistance to synthetic and traditional
biological insecticides (Salgado 1997, 1998). Moreover,
spinosad is classified by the US Environmental Protection
Agency as an environmentally and toxicologically reduced
risk material (Saunders and Bret 1997).
In insects, the mode of action of spinosad is associated
with the excitation of the insect nervous system (Salgado
1998). Although the exact site of action of spinosyns is still
under investigation, spinosad uniquely alters the function of
nicotinic and GABA-gated ion channels at the synapse
between nerve cells. However, spinosad does not interact
with known binding sites for other nicotinic or GABA
insecticides such as neonicotinoids, fiproles, avermectins,
and cyclodienes. These data indicate that spinosad acts
through a unique insecticidal mechanism.
Spinosad is a novel insecticide produced from a
family of natural products derived from fermentation of
the actinomycete Saccharopolyspora spinosa (Snyder et
al. 2007). These two bacterial biocides have been
evaluated in various formulations against mosquito vec-
tors worldwide (Lacey and Lacey 1990). Bacillus thur-
ingiensis produces four key insecticidal proteins, and
Bacillus sphaericus produces a single binary toxin
effective against JE vector (Federici et al. 2003). B.
sphaericus and B. thuringiensis H-14 showed larvicidal
activity up to 9199% against Culex tritaeniorhynchus and
Anopheles subpictus; however, activity did not subsist
beyond a few days (Kramer 1984). B. thuringiensis H-14
showed 100% reduction with doses of 27105 spores per
milliliter, but first and second instars reappeared 3 days
after application (Balaraman et al. 1983).
Bond et al. (2004) reported that the naturally derived
insecticide spinosad is highly toxic to Aedes and Anopheles
mosquito larvae. Cetin et al. (2005) worked on the
Evaluation of the naturally derived insecticide spinosad
against Culex pipiens L. (Diptera: Culicidae) larvae in septic
tank water in Antalya. Additional studies have reported the
larvicidal properties of spinosad in this and other mosquito
species (Liu et al. 2004a, 2004b; Cetin et al. 2005a; Darriet
et al. 2005; Darriet and Corbel 2006; Romi et al. 2006), or as
an adulticide in a sugar bait formulation (Muller and Schlein
2006).
During the present study, an attempt was made to
establish the larvicidal and pupicidal properties of methanol
leaf crude extract of C. papaya and bacterial insecticide,
spinosad against chikungunya vector, A. aegypti as target
species.
671
Materials and methods
Collection of eggs and maintenance of larvae
The eggs of A. aegypti were collected from National
Centre for Disease Control (NCDC) field station of
Mettupalayam, Tamil Nadu, India, using anO 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
fed with 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
transferred to plastic containers (1212 cm) containing
500 ml of water with the help of a dipper. The plastic jars
were kept in a 909090-cm mosquito cage for adult
emergence. Mosquito larvae were maintained at 27C+2C,
7585% RH under a photoperiod of 14 L/10 D. A 10%
sugar solution was provided for a period of 3 days before
blood feeding.
Blood feeding of adult A. aegypti
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
oviposition substrates.
Plant bioassay
C. papaya was collected in and around Bharathiar
University, Coimbatore, India. The voucher specimen
has been deposited at the Zoology Department, Bhar-
athiar University, Coimbatore, Tamil Nadu, India. C.
papaya leaf was washed with tap water and shade-dried at
room temperature. An electrical blender powdered the
dried plant materials (leaves). The powder (500 g) of the
leaf was extracted with 1.5 l of organic solvents of
methanol for 8 h using a Soxhlet apparatus (Vogel 1978).
The extracts were filtered through a Buchner funnel with
Whatman number 1 filter paper. The crude plant extracts
were evaporated to dryness in rotary vacuum evaporator.
One gram of the plant residue was dissolved in 100 ml of
acetone (stock solution) and considered as 1% stock
solution. From this stock solution, different concentra-
tions were prepared ranging from 100 to 500 ppm,
respectively.
672
Microbial bioassay
Spinosad was obtained from T-Stanes & Company
Limited, Research and Development Centre, Coimbatore,
Tamil Nadu, India. The required quantity of spinosad
was thoroughly mixed with distilled water to prepare at
various concentrations ranging from 20 to 100 ppm,
respectively.
Larval/pupal toxicity test
Laboratory colonies of mosquito larvae/pupae were used for
the larvicidal/pupicidal activity. Twenty-five numbers of I to
IV instar larvae and pupae were introduced into 500-ml glass
beaker containing 249 ml of dechlorinated water and 1 ml of
desired concentrations of plant extract, and spinosad were
added. Larval food was given for the test larvae. At each tested
concentration, two to five trials were made, and each trial
consisted of three replicates. The control was set up by mixing
1 ml of acetone with 249 ml of dechlorinated water. The larvae
and pupae which were exposed to dechlorinated water without
acetone served as control. The control mortalities were
corrected by using Abbott's formula (Abbott's 1925). The
LC50 and LC90 were calculated from toxicity data by using
probit analysis (Finney 1971).
Corrected mortality
Observed mortality in treatment  Observed mortality in control
 100
100  Control mortality
Number of dead larvae=pupae
Percentage mortality  100
Number of larvae=pupae introduced
Statistical analysis
All data were subjected to analysis of variance; the means
were separated using Duncan's multiple range tests by
Alder and Rossler (1977). The average larval mortality data
were subjected to probit analysis, for calculating LC50 and
LC90, values were calculated by using the Finney (1971)
method. SPSS software package 9.0 version was used.
Results with P<0.05 were considered to be statistically
significant.
Results
Larval and pupal mortality of A. aegypti after the treatment
of methanolic extract of C. papaya leaf was observed.
Table 1 provides the larval and pupal mortality of A. aegypti
(I to IV instars) after the treatment of A. aegypti at different
Parasitol Res (2012) 110:669678
concentrations (100 to 500 ppm). Thirty-four percent
mortality was noted at I instar larvae by the treatment of
C. papaya at 100 ppm, whereas it has been increased to
92% at 500 ppm of C. papaya leaf extract treatment.
Similar trend has been noted for all the instars of A. aegypti
at different concentration of C. papaya treatment. The LC50
and LC90 values were represented as follows: LC50 value of
I instar was 227.15 ppm, II instar was 277.16 ppm, III
instar was 335.99 ppm, and IV instar was 375.88 ppm,
respectively. The LC90 value of I instar was 536.36 ppm, II
instar was 593.90 ppm, III instar was 679.51 ppm, and IV
instar was 715.75 ppm, respectively. The LC50 value of
pupae was 440.65 ppm, and the LC90 value of pupae was
796.59 ppm, respectively.
Table 2 illustrates the larval and pupal mortality of A.
aegypti (I to IV instars) after the treatment of spinosad at
different concentrations (20100 ppm). Thirty-one per-
cent mortality was noted at I instar larvae by the
treatment of spinosad at 20 ppm, whereas it has been
increased to 87% at 100 ppm of spinosad treatment and
12% mortality was noted at pupae by the treatment of
spinosad at 20 ppm and it has been increased to 64% at
100 ppm. Similar trend has been noted for all the instars
of A. aegypti at different concentrations of spinosad
treatment. The LC50 and LC90 values were represented
as follows: LC50 value of I instar was 51.76 ppm, II instar
was 61.87 ppm, III instar was 74.07 ppm, and IV instar
was 82.18 ppm, respectively. The LC90 value of I instar
was 117.60 ppm, II instar was 139.27 ppm, III instar was
149.03 ppm, and IV instar was 155.50 ppm, respectively.
The LC50 value of pupae was 93.44 ppm and the LC90
value of pupae was 162.66 ppm, respectively.
Table 3 shows the considerable larval and pupal
mortality after the combined treatment of spinosad and
C. papaya leaf of methanolic extract for all the larval
instars and pupae. The concentration at 50+100 ppm
combined treatment of spinosad and C. papaya for I instar
larval mortality was 95%, respectively. The LC50 and
LC90 values were represented as follows: LC50 value of I
instar was 55.77 ppm, II instar was 65.77 ppm, III instar
was 76.36 ppm, and IV instar was 92.78 ppm. The LC90
value of I instar was 151.17 ppm, II instar was 175.79, III
instar was 190.98 ppm, and IV instar was 202.94 ppm. The
LC50 value of pupae was 107.62 ppm, and the LC90 value
of pupae was 222.92 ppm, respectively.
Discussion
Dengue is an arboviral disease mainly transmitted by the
mosquito A. aegypti, and in the last years has become a
major international public health concern (WHO 1998). It is
found in tropical and subtropical regions around the
Parasitol Res (2012) 110:669678 673

Table 1 Larval and pupal toxicity effect of C. papaya leaf extract against the chikungunya vector, A. aegypti

Mosquito larval Larval and pupal LC50 (LC90) 95% Confidence x2 (df=4)
instars and pupae mortality limit

Concentration of LFL UFL

C. papaya (ppm)

100 200 300 400 500 LC50 (LC90) LC50 (LC90)

I 34a 45a 56a 73a 92a 227.15280 (536.36209) 192.98940 (484.34574) 256.16186 (612.86015) 5.015*
II 29b 35b 47b 68b 86b 277.16385 (593.90556) 246.74353 (534.82633) 306.10427 (681.63126) 5.006*
III 22c 31b 40c 54c 79c 335.99208 (679.51541) 304.96606 (604.56073) 370.39636 (795.48488) 4.570*
IV 18cd 26c 34d 47d 75c 375.88635 (715.75632) 306.16292 (567.78724) 490.40852 (1,150.40125) 5.729*
Pupa 14d 20d 27e 35e 67d 440.65630 (796.59968) 350.71643 (597.86625) 701.11584 (1,683.05935) 7.991*

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT
Control-Nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05 level

world, predominantly in urban and semi-urban regions.
The WHO published a global map of the distribution of
the dengue epidemic activity during the year 2006 that
shows whole India in red color. More than 50 million
people are at risk of dengue virus exposure worldwide.
Annually, there are two million infections, 500,000
cases of dengue hemorrhagic fever, and 12,000 deaths
(Guha-Sapir and Schimmer 2005). Conventional control
of A. aegypti is based on treatment of water containers
with larvicides and on the use of adulticides (Gubler and
Clark 1995; Gubler 2004).
The main objective of the current study is to investigate
the potential of C. papaya leaves extract against dengue
fever. The secondary metabolite of plant origins makes up a
vast repository compounds with a wide range of biological
activities. There have been many reports of higher plant
extracts possessing relatively good potential to inhibit
viruses (Van Den Berghe 1978). Laboratory bioassay of
latex from the unripe fruits of C. papaya was carried out
against A. stephensi, and the LC50 and LC90 value was
0.013% and 0.062%, respectively (Thomas et al. 2004).
Roark (1947) described approximately 1,200 plant spe-
cies, whilst Sukumar et al. (1991) listed and discussed 344
plant species that exhibited mosquitocidal activity. Shaalan et
al. (2005) reviewed the current state of knowledge on
larvicidal plant species and listed the growth and reproduc-
tion inhibiting phytochemicals, botanical ovicides, synergis-
tic, additive and antagonistic joint action effects of botanical
mixtures, residual capacity and effects on nontarget organ-
isms, and appearance of resistance. Usually, it has been
found that secondary metabolites produced by plants are
responsible for their chemical defense and toxicity to other
animals. Several secondary metabolites such as steroids
(Ghosh et al. 2008; Chowdhury et al. 2008; Rahuman et al.
2008; Zolotar et al. 2002), phenolics (Tripathi and Rathore
2001), essential oils (Amer and Mehlhorn 2006).

Table 2 Larval and pupal toxicity effect of bacterial insecticide, spinosad against the chikungunya vector, A. aegypti

Mosquito larval Larval and pupal LC50 (LC90) 95% Confidence limit x2 (df=4)
instars and pupae mortality

Concentration of spinosad (ppm) LFL UFL

20 40 60 80 100 LC50 (LC90) LC50 (LC90)

I 31a 39a 52a 68a 87a 51.76882 (117.60759) 45.15904 (105.51486) 57.74529 (135.82611) 3.563*
II 26b 33b 44b 55b 80b 61.87610 (139.27069) 44.34864 (108.38719) 81.06919 (240.91736) 5.534*
III 20c 29b 37c 48c 73c 74.07166 (149.03313) 67.19039 (130.75840) 82.51930 (178.61302) 3.931*
IV 17c 23c 31d 42d 69c 82.18527 (155.50594) 74.89372 (136.34454) 91.89465 (186.53804) 5.135*
Pupa 12d 16d 24e 30e 64d 93.44808 (162.66591) 73.60570 (119.98824) 167.99669 (397.58642) 9.474*

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT
Control-Nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05 level
674 Parasitol Res (2012) 110:669678

Table 3 Combined treatment of larval and pupal toxicity effect of C. papaya leaf extract and bacterial insecticide, spinosad against the
chickugunya vector, A. aegypti

Mosquito larval Larval and LC50 (LC90) 95% Confidence limit x2 (df=4)
instars and pupae pupal mortality

Concentration of C. papaya LFL LC50 (LC90) UFL LC50 (LC90)

(ppm)+Spinosad (ppm)

20+10 40+20 60+30 80+40 100+50

I 42a 51a 60a 78a 95a 55.77193 (151.17382) 6.41336 (119.41368) 78.84710 (253.41303) 7.563*
II 39a 45b 56b 70b 89b 65.77360 (175.79516) 53.04977 (155.84897) 76.00133 (207.29224) 5.253*
III 30b 41bc 52b 65b 83c 76.36812 (190.98989) 64.59055 (168.32860) 86.63494 (227.48104) 2.813*
IV 24c 37cd 46c 59c 78c 92.78842 (202.94203) 82.76361 (179.35220) 103.12543 (240.36802) 1.545*
c
Pupa 19 32d 40d 55c 69d 107.62874 (222.92774) 97.21988 (195.14688) 120.05786 (268.31690) 0.486*

Within a column means followed by the same letter(s) are not significantly different at 5% level by DMRT
Control-Nil mortality, LFL lower fiducidal limit, UFL upper fiducidal limit, x2 Chi-square value, df degrees of freedom
*P<0.05 level

Sharma et al. (2005) reported that the acetone extract of
Nerium indicum and Thenus orientalis have been studied
with LC 50 values of 200.87, 127.53, 209.00, and
155.97 ppm against III instar larvae of A. stephensi and
C. quinquefasciatus, respectively. Earlier authors reported
that the methanol leaf extracts of Vitex negundo, Vitex
trifolia, Vitex peduncularis, and Vitex altissima were used
for larvicidal assay with LC50 values of 212.57, 41.41,
76.28, and 128.04 ppm, respectively, against the early
fourth-instar larvae of C. quinquefasciatus (Kannathasan et
al. 2007). The larvicidal activity of the essential oil aqueous
solutions of the stalks and leaves of Croton argyrophyl-
loides, Croton nepetaefolius, Croton sonderianus, and
Croton zehntneri showed 100% mortality at 50 ml against
A. aegypti (Lima et al. 2006); Morais et al. (2006) also
reported that the main components methyleugenol and
alpha-copaene for C. nepetaefolius (LC50 of 84 ppm);
alpha-pinene and beta-pinene for C. argyrophyloides (LC50
of 102 ppm); and alpha-pinene, beta-phelandrene, and
transcaryophyllene for C. sonderianus (LC50 of 104 ppm)
and Croton zenhtneri exhibited higher larvicidal activity
with an LC50 of 28 ppm against A. aegypti. The toxicity of
Euphorbia milii molluscicidal latex and niclosamide
showed toxic affect to Anopheles albitarsis, A. aegypti,
and Aedes fluviatilis larvae (Filho and Paumgartten 2000).
Likewise, the crude extracts of a few indigenous plants
showed moderate larvicidal effects against larvae of Culex
quinquefasciatus Say with LC50 value ranging from 41.75
to 709.51 ppm (Rahuman et al. 2009a).
Recently, studies stimulated that the larval and pupal
mortality of A. stephensi after the treatment of methanolic
extract of C. inerme leaf extract is shown; results of 22%
mortality was noted at I instar larvae by the treatment at
20 ppm, whereas it has been increased to 81% at 100 ppm
of C. inerme leaf extract of larval and pupal mortality of A.
stephensi (I to IV instars) after the treatment of methanolic
extract of A. ilicifolius at different concentrations (20
100 ppm). Twenty-three percent mortality was noted at I
instar larvae by the treatment of A. ilicifolius at 20 ppm,
whereas it has been increased to 89% at 100 ppm of A.
ilicifolius leaf extract treatment (Kovendan and Murugan
2011). Kovendan et al. (2011a, b) recently have reported
that the leaf extract of methanol Jatropha curcas against C.
quinquefasciatus and Leucas aspera leaf extract against A.
stephensi, respectively.
The bio-control potentiality of crude extracts of the three
plants, C. papaya, M. paniculata, and C. collinus against C.
quinquefasciatus have been well-established in the
laboratory condition (Rawani et al. 2009). The highest
mortality was recorded in C. papaya seed extract. The
phytochemical analysis of the plant extracts reveals the
presence of several bioactive secondary metabolites that
singly or in combinations may be responsible for the larval
toxicity. As no mortality occurs in the non-target organisms
(invertebrates), it can be assumed that all the plant extracts are
safe to use in the aquatic ecosystem, though some toxicity of
C. collinus in higher vertebrates have been reported
(Sarathchandra and Balakrishnamoorthy 1998; Eswarappa
and Benjamin 2007). In conclusion, the crude extracts of C.
papaya, M. paniculata, and C. collinus can be recommended
in large-scale field trials and can be effectively used as potent
larvicides in mosquito control programs. During present
study, C. papaya methanolic extract was of considerable and
good larvicidal and pupicidal properties against the
chikungunya vector, A. aegypti.
Cisneros et al. (2002) said that spinosad acts as a
stomach and contact poison and degrades rapidly in the
environment. Earlier, Bond et al. (2004) have reported that
the spinosad was most effective at the lowest concentrations
(0.0240.025). Romi et al. (2006) studied the efficacy of a
Parasitol Res (2012) 110:669678
spinosad-based product (laser 4.8% emulsifiable concentrate)
which was evaluated in laboratory bioassays against
laboratory-reared mosquito strains of three species of medical
importance: A. aegypti, A. stephensi, and C. pipiens.
Spinosad was particularly effective against larval Aedes and
Culex, with a less marked activity against Anophelines (24-
h median lethal concentration = 0.0096, 0.0064, and
0.039 mg/l, respectively), showing a persistence of the
insecticide action of about 6 weeks in laboratory containers.
However, it was clear that spinosad solutions placed in
warm, sunny locations lost toxicity tenfold faster than
solutions placed in the shade. Because A. aegypti preferen-
tially oviposits in shaded habitats (Fay and Eliason 1966;
Vezzani et al. 2005), the ability of spinosad to persist for
weeks or months in the shade favors the suppression of
mosquito development for periods that extend over the
annual peaks of vectorial activity and that often coincide
with seasonal fluctuations in rainfall in tropical regions.
The insignificant mammalian toxicity and favorable
environmental profile of spinosad, involving degradation
by photolysis and microbial action (Cleveland et al.
2002; Thompson et al. 2002; Liu and Li 2004), means that
bioaccumulation and related ecological problems that arise
from persistent xenobiotic compounds are highly unlikely
for this product.
High concentrations of organophosphate and pyrethroid
insecticides tend to be deterrent for oviposition (Moore
1977; Verma 1986), whereas other compounds, such as
methoprene or granular formulations of temephos, are not
repellent (Mather and DeFoliart 1983; Beehler and Mulla
1993; Pates and Curtis 2005). In our study, a weak but
significant attraction to visit cups containing spinosad was
observed at a concentration of 20 ppm but not at 5 ppm.
Spinosad has a distinctive aroma of damp earth, characteristic
of the presence of actinomycetes that may have proved
attractive to gravid females. However, it did not result in an
increase in the number of eggs laid in the spinosad treatments,
or any other of the treatments that we tested. This finding
could have been influenced by the response of A. aegypti to
conspecific eggs (Allan and Kline 1998) or by the skip
oviposition behavior shown by some, but not all populations,
of this species (Corbet and Chadee 1993; Harrington and
Edman 2001), and the limited possibility to disperse eggs
over various oviposition sites in our caged experiments.
Perez et al. (2007) have reported that the naturally
derived insecticide spinosad is a reduced-risk material that
is neurotoxic to Diptera. The 24-h 50% lethal concentration
by laboratory bioassay in third instars of A. aegypti (L.)
(Diptera: Culicidae) (Rockefeller strain) was estimated at
0.026 ppm. Water containers treated with 1 or 5 ppm
spinosad suspension concentrate (Tracer, DowAgrosciences)
were as effective in preventing the development of Aedes spp.
(mostly A. aegypti) as temephos granules during both trials,
675
whereas the bacterial insecticide VectoBac 12AS per-
formed poorly. The half-life of aqueous solutions of
spinosad (10 ppm) placed in a warm sunny location was
2.1 days, compared with 24.5 days for solutions in a
shaded location. Spinosad was as effective as temephos
granules in eliminating the immature stages of Aedes
spp., mostly A. aegypti, in an urban cemetery during the
wet and dry seasons in southern Mexico. Persistence and
oviposition response studies indicated that spinosad could
retain its insecticidal properties for periods of several
months in shaded conditions preferred by A. aegypti, and
it was not repellent for mosquito oviposition. The
combination of the toxicological properties and favorable
environmental profile means that spinosad deserves
detailed evaluation as a mosquito larvicide in domestic
and urban vector control programs.
In earlier study, the microbial pesticide spinosad against
the malarial vector A. stephensi Liston showed 85%
mortality (Aarthi and Murugan 2010). The observed
mortality rate suggests that the above extract can be used
as biopesticides. The LC50 of second, third, and fourth-
instar larvae of A. stephensi were 0.276%, 0.285%, and
0.305%, respectively. In the present study, spinosad
treatment reduced the larval and pupal properties of
microbial insecticides development of growth control.
Garcia and Desrochers (1979) observed appreciable
mortality only with high concentrations (1107 cells per
milliliter) of B. thuringiensis var. israelensis. The biocide at
1 to 10 Kg/ha (0.252.5 ppm) caused 18% to 88% mortality
of midges during a 4-week evaluation period. Younger
instars are more susceptible than older ones as shown by C.
quinquefasciatus. Exposure periods longer than 48 h in the
laboratory may produce better activity results of the B.
thuringiensis var israelensis formulations against the
midges' species (Ali 1981). Similarly, the alternative use
of plant extracts as an additive to B. thuringiensis var.
israelensis may be a promising approach because larval
feeding and subsequent defoliation would be reduced
greatly without interference with bacterial activity.
Ludlum et al. (1991) have reported that aromatic com-
pounds and plant allelochemicals increase B. thuringiensis
var. israelensis activity. The addition of B. thuringiensis var.
israelensis with the plant extracts had an adverse effect upon
the larval mortality. When combined with plant extracts, B.
thuringiensis var. israelensis increased the percentage of
larval mortality and decreased the time to kill when
compared with treatment containing only B. thuringiensis
var. israelensis. The addition of B. thuringiensis var. israel-
ensis with plant extracts caused a significant mortality due to
the avoidance of treated diet and may be due to increased
toxicity (Gould et al. 1991). Since the discovery of the agent
and its lethal effects against species of Anopheles, Aedes,
Culex, Ochlerotatus, and Uranotaenia larvae by Goldberg
676
and Margalit (1977), Kovendal et al. (2011) recently
reported that B. thuringiensis israelensis against the first
to fourth-instar larvae of values LC50 =9.332%, 9.832%,
10.212%, 10.622%, and LC 90 = 15.225%, 15.508%,
15.887%, and 15.986% larvae of C. quinquefasciatus,
respectively.
In the present study, 31% mortality was noted at I instar
larvae by the treatment of spinosad at 20 ppm, whereas it
has been increased to 87% at 100 ppm of spinosad
treatment and 12% mortality was noted at pupae by the
treatment of spinosad at 20 ppm, and it has been increased
to 64% at 100 ppm. Similar trend has been noted for all the
instars of A. aegypti at different concentrations of spinosad
treatment. LC50 value of I instar was 51.76 ppm, II instar
was 61.87 ppm, III instar was 74.07 ppm, and IV instar was
82.18 ppm, respectively. The LC90 value of I instar was
117.60 ppm, II instar was 139.27 ppm, III instar was
149.03 ppm, and IV instar was 155.50 ppm, respectively.
The concentration at 50+100 ppm combined treatment of
spinosad and C. papaya for I instar larval mortality was
95%, respectively
The finding of the present investigation revealed that the
leaf extract of C. papaya and bacterial insecticide, spinosad
has good larvicidal and pupicidal properties of against
chikungunya vector, A. aegypti as target species of vector
control programs.
Acknowledgments The authors are thankful to the Department of
Science and Technology (DST), New Delhi, India and Tamil Nadu
State Council for Science and Technology (TNSCST), Chennai,
Tamil Nadu for providing financial support for the present work.
The authors are grateful to Dr. K. Sasikala, Professor and Head,
Department of Zoology, Bharathiar University for the laboratory
facilities provided.
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