International Journal of Pure and Applied Zoology ISSN (Print) : 2320-9577

Volume 2, Issue 2, pp: 71-83, 2014 ISSN (Online): 2320-9585

©2013 Rishan Publications http://www.ijpaz.com
Research Article

LARVICIDAL, REPELLENT AND SMOKE TOXICITY EFFECT OF NEEM

PRODUCTS AGAINST MALARIALVECTOR, ANOPHELES STEPHENSI

K. Murugan1*, G. James Pitchai1, P. Madhiyazhagan1, T. Nataraj1,

A. Nareshkumar2, Jiang-Shiou Hwang3, R. Chandrasekar4, M. Nicoletti5,
A. Amsath6 and Ranjeet Bhagooli7
*1
Division of Entomology, Department of Zoology, School of Life Sciences, Bharathiar
University, Coimbatore-641 046, Tamil Nadu, India.
2
Division of Entomology, Department of Zoology, Periyar University, Salem, India.
3
Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, 202.
5
Department of Biochemistry and Molecular Biophysics, 238 Burt Hall, Kansas State
University, Manhattan 66506, KS, USA.
5
Department of Environmental Biology, University Sapienza of Rome, P.le A., Moro,
5-00185, Italy.
6
PG and Research Department of Zoology, Khadir Mohideen College, Adirampattinam-
614 701, Tamil Nadu, India.
7
Department of Biosciences, Faculty of Science, University of Mauritius, Reduit- 80837,
Mauritius.
Article History: Received 2nd January 2014; Accepted 25th March 2014; Published online 3rd April 2014.

ABSTRACT
Vector control plays a key role in prevention and control of major vector-borne diseases and often constitutes
the first line of activity in case of epidemics of vector-borne diseases, and particularly, malaria. Chemical
control (use of pesticides) is still the most important element in the integrated approach to vector control. But
they are non-selective and harmful to other beneficial organisms. Some of the insecticides are carcinogenic
agents and are positively dangerous and even carried through the food chain which in turn affects the non-target
organism. In view of the above, the uses of biologically-active plant materials with anti-mosquitocidal
properties and ecofriendly-biopesticides are attracted in recent years, because of their biodegradable nature and
being relatively safer to human and other non-target organism in the environment. The present paper is to
investigate on the larvicidal, pupicidal, smoke repellency effect of neem products against malarial vector,
Anopheles stephensi. Six neem limonoids (purity>99%), namely azadiractin, salannin, deacetylgedunin,
gedunin, 17-hydroxyazadiradione and deacetylnimbin were sent from Central Research Laboratories, Taiyo
Kagaku Co Ltd., Japan. Larvicidal bioassays were conducted at the laboratory with neem limonoids the lethal
concentrations (LC50, LC90) were worked out by Abottts’ formula. Repellency bioassay was done by human
volunteers by using neem oil which was procured from the Local Neem Oil Mill, Kalverrampalayam,
Coimbatore-641 046, India. Smoke toxicity was performed on the adult female mosquitoes at the laboratory by
using neem seed kernel power and it was collected from the Bharathiar University Campus, Coimbaotore-641
046, India. The neem products also had significant larvicidal activity. The larval mortality was dose dependant.
The LC50 and LC90values of Azadirachtin treatment at 0.50, 1.0 and 1.5 ppm concentrations was 0.299% and
1.061%, respectively. After treatment of neem oil at 0.50, 1.0 and 1.5 ppm concentrations were 0.503 and 1.324,
respectively. After the treatment of salanin, 17-Hydroxyazadiradione, Deacetyl gedunin, Gedunin and Dacetyl
nimbin the LC50 and LC90 values were increased when compare to Azadirachtin. There was significant repellent
activity after the treatment of neem products at three different concentrations (0.2, 0.4, and 0.6). Neem oil had
higher repellent activity (<300 minutes at 0.6 concentration), followed by Gedunin which showed less activity
(<200 minutes at 0.6 concentration). However, the ethanol applied arm served as control which provided
maximum 7.0 minutes repellency. The leaves and pods were also tested smoke repellency bioassay. Smoke

*Corresponding author e-mail: kmvvkg@gmail.com, Mobile: +91 9894832849

Murugan et al. Int. J. Pure Appl. Zool., 2(2): 71-83, 2014

emerged from the leaves and pods greater knock down effect were evident and percentage of repellency on leaf
59%, pod 53% and with commercial coil as positive control which showed 65% repellency. Moreover, smoke
exposed larvae laid minimum number of eggs and hatchability also affected. From this study it has been
concluded that based on the larvicidal, smoke repellent properties neem products showed mosquitocidal
properties. Hence, it can be concluded that neem as effective biopesticides for the Integrated Vector Control
Program. Moreover, neem is indigenous, medicinal importance and much of social relevance to people and
traditional knowledge of our country.
Keywords: Neem products, larvicide, repellent, smoke toxicity, malarial vector.

INTRODUCTION reduced fecundity, moulting disorders,

morphogenetic defects, and changes of behavior
Mosquitoes are the prime vectors responsible for
(Schmidt et al., 1998; Abou Fakhr Hammad et
transmission of diseases to more than 70 billion
al., 2001; Gajmer et al., 2002; Banchio et al.,
people annually worldwide. As per the reports of 2003; Wandscheer et al., 2004). Protection from
World Health Organization, malaria alone kills arthropod bites is best achieved by avoiding or
30 million people annually. Mosquitoes transmit destroying infested habitats, wearing protective
the arbo-viruses which cause yellow fever, clothing, and using insect repellents. Repellents
dengue hemorrhagic fever, epidemic that are currently available in the market are
polyarthritis, and several forms of encephalitis either synthetic chemicals, such as N, N-diethyl-
eg. Bancroftian filariasis which is caused by a meta-toluamide (DEET), picaridin, ethyl butyl
nematode transmitted by mosquito bite (Mark acetyl aminoproprionate or plant derived
and Fradin, 1998). Anopheles stephensi Liston chemicals such as Citronella oil, oil of lemon
(Diptera: Culicidae) is a major vector in India as eucalyptus. These insect repellent products are
well as in some of the West Asian countries and available in various types of formulation as
has been shown to be directly responsible for sprays, sticks, foams, and lotions (Fradin and
about 40-50% of the annual malarial incidence Day 2002). Mosquito control and personal
(Curtis, 1994; Collins and Paskewitz, 1995). protection from mosquito bites are currently the
Globally, malaria kills 3 million people each most important measures to control these
year, including 1 child every 30 seconds (Shell, diseases. The use of repellents is an obvious
1997). Plant product has been used by practical and economical means of preventing
traditionally human communities many part of the transmission of these diseases to humans.
the world against the vectors and species of The most common mosquito repellent
insects. The phyto-chemicals derived from plant formulations available on the market contain deet
sources can act as larvicides, insect growth (N, N-diethyl-3-methylbenzamide), which has
shown excellent repellency against mosquitoes
regulators, repellent and ovipositional attractants
and other biting insects (Yap 1986, Coleman
and have deterrent activities observed by many
et al., 1993).
researchers (Babu and Murugan, 1998).
Recently, extracts of several plants including
In India, bioactivity of 344 botanical agents neem (Azadirachta indica A. Juss), Citronella
(Sukumar et al., 1991) and active chemicals from grass (Cymbopogan nardus Rendle), basil
neem were tested against mosquitoes (Ocimum basilicum L., Ocimum gratissimum L.,
(Schmutterer 1988, 1990). Melia azedarach L. Ocimum americanum L.), clove (Syzygium
and Azadirachta indica (Sapindales: Meliaceae), aromaticum L.), prickly straggler (Solanum
commonly known as Chinaberry or Persian lilac trilobatum L.), musk basil (Moschosma
tree, are deciduous trees that are native to polystachyum L.) and thyme (Thymus vulgaris
northwestern India; and have long been
L.) have been studied as possible mosquito
recognized for their insecticidal properties. These
trees typically grow in the tropical and repellents (Chokechaijaroenporn et al., 1994;
subtropical parts of Asia, but now-a-days they Suwonkerd & Tantrarongraj, 1994; Rajkumar &
are also cultivated in other warm regions of the Jebanesan, 2004, 2005). Neem trees,
world because of their considerable climatic (Azadirachta indica) native of India, belonging
tolerance. Fruit extracts of Melia azedarach and to family Meliaceae are fast growing evergreen
Azadirachta indica elicit a variety of effects in trees ranging in height from 12 – 24 m. They are
insects such as antifeedant, growth retardation, widespread in tropical and subtropical regions of

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Murugan et al. Int. J. Pure Appl. Zool., 2(2): 71-83, 2014

the world, including semi-arid and wet- tropical and tannins, aliphatic compounds, etc. The
regions (National Research Council, 1992). details of the chemistry of various compounds
Neem seeds contain approximately 99 falling under these groups have already been
biologically active compounds of which reviewed (Kraus, 1995, Devakumar and
azadirachtin, nimbin, nimbidin and nimbolides SukhDev, 1996).
are major molecules. Many of these derived In this group of neem products are capable of
products have antifeedancy, ovicidal activity, inflicting multiple effects on insects such as
fecundity suppression besides insect growth antifeedant, growth regulation, fecundity,
regulation and repellency against insects (Isman, suppression and sterilization, oviposition,
2006, Schmutterer, 2002). repellency or attractancy and changes in
biological fitness. Neem (Azadirachtica indica
Neem products have low toxicity to birds,
A.Juss) is parahaps the most useful traditional
fish and mammals and are less likely to induce pesticidal plant in India. Each part of the neem
resistance due to their multiple mode of action on tree has insecticidal properties (Murugan et al.,
insects. In addition to this, insect growth 1995; Murugan and Jeyabalan, 1995; Murugan et
regulatory activity of neem weakens the cuticle al.,1999).Nimbidin, a major crude bitter principle
defence system of the larvae causing easy extracted from the oil of seed kernels of A. indica
penetration of pathogenic organisms into insect demonstrated several biological activities. From
system. Azadirachtin, a biologically active this crude principle some tetranortriterpenes,
compound has been promoted as a new including nimbin, nimbinin, nimbidinin,
insecticide that is considered more eco- friendly nimbolide and nimbidic acid have been isolated
than synthetic insecticides. The pesticidal (Siddiqui, 1942, Mitra et al., 1971).The
efficacy, environmental safety and public spermicidal activity of nimbidin and nimbin (1)
acceptability of neem and its products for control was reported in rats and human as early as 1959
of crop pests has led to its adoption into various (Sharma and Saksena, 1959). Nimbidin also
mosquito control programmes (Su and Mulla, demonstrated antifungal activity by inhibiting the
1998, Su and Mulla, 1998). Chemical growth of Tinea rubrum (MurthySirsi, 1958). .
investigation on the products of the neem tree Nimbolide (2) has been shown to exert
was extensively undertaken in the middle of the antimalarial activity by inhibiting the growth of
twentieth century. Since the early report by Plasmodium falciparum (Rochanakij, et al.,
(Siddiqui, 1942) in 1942 on the isolation of 1985, Khalid, et al., 1989). Nimbolide also
nimbin, the first bitter compound isolated from shows antibacterial activityagainst S. aureus and
neem oil, more than 135 compounds have been S. coagulase (Rojanapo, et al., 1985). Gedunin
isolated from different parts of neem and several (3), isolated from neem seed oil has beenreported
reviews have also been published on the to possess both antifungal (Rao, et al., 1986) and
chemistry and structural diversity of these antimalarial(Khalid, et al., 1989) activities.
compounds (Koul et al., 1990, Chatterjee and Azadirachtin (4), highly oxygenated C-
Pakrashi, 1994, Mitra, 1963, Warthen, 1979, secomeliacins isolated from neem seed and
Taylor, 1984, Champagne, et al., 1992, Kraus, having strong antifeedant activity (Kraus, 1995,
1995, Devakumar and Sukh Dev, 1996, Govindachari, 1992, Kirtikar and Basu, 1975).
Govindachari, 1992). The compounds have been Repellent action of neem oil in the form of
divided into two major classes: isoprenoids and mats (Sharma et al., 1993) and neem cream (Dua
others (Kraus, 1995). The isoprenoids include et al., 1995) have been evaluated against
diterpenoids and triterpenoids containing mosquitoes. Benzene and methanol extracts of
protomeliacins, limonoids, azadirone and its Artemisia vulgaris have been reported to have
derivatives, gedunin and its derivatives, vilasinin repellent activity against Ae. aegypti (Yit et al.,
type of compounds and Csecomeliacins such as 1985). Two types of botanical insecticides can be
nimbin, salanin and azadirachtin. The obtained from seeds of the Indian neem tree,
nonisoprenoids include proteins (amino acids) Azadirachta indica (Meliaceae) (Schmutterer,
and carbohydrates (polysaccharides), sulphurous 2002). Neem oil, obtained by cold-pressing
compounds, polyphenolics such as flavonoids seeds, can be effective against soft-bodied insects
and their glycosides, dihydrochalcone, coumarin and mites but is also useful in the management of
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phytopathogens. Apart from the physical effects cups used for any experimental purpose during
of neem oil on pests and fungi, disulfides in the the present study were kept closed with muslin
oil likely contribute to the bioactivity of this cloth for preventing contamination through
material. More highly valued than neem oil are foreign mosquitoes.
medium-polarity extracts of the seed residues
3. Maintenance of pupae and adult
after removal of the oil, as these extracts contain
the complex triterpene azadirachtin. Neem seeds The pupae were collected from culture trays and
actually contain more than dozen azadirachtin were transferred to glass beakers containing 500
analogs, but the major form is azadirachtin and ml of water with help of a sucker. The glass
the remaining minor analogs likely contribute beaker containing pupae was then kept in 90 x 90
little to overall efficacy of the extract. Seed x 90 cm size mosquito cage for adult emergence.
extracts include considerable quantities of other The cage was made up of wooden frames
triterpenoids, notably salannin, nimbin, and and covered with polythene sheets on four sides
derivatives thereof. The role of these other (two laterals, one back and other one upper) and
natural substances has been controversial, but the front part was covered with a muslin cloth.
most evidence points to azadirachtin as the most The bottom of the cage was fitted with strong
important activeprinciple (Isman et al., 1996). cardboard. The freshly emerged adults were
(Neem oil Limonoids) is a trade marked natural
maintained 27 ± 2°C, 75 - 85% RH, under 14L:
extract from Sabinsa Corporation, obtained from
10D photoperiod cycles. The adults were fed
cold pressed neem seed oil and standardized to
with 10% sugar solution for a period of three
contain not less than 50% Total Limonoids and
days before they were provided an animal for
1000 ppm Azadirachtin. Potential cosmeceutical
blood feeding.
applications include antibacterial, antifungal,
antiparasitic, insect repellant, anti-pediculosis 4. Blood feeding of adult Anopheles stephensi
formulations for topical use in skin and hair care. and egg laying
(Neem Oil Limonoids: Product Overview, 2007). The females were fed by hand every alternate
The present paper is to investigate on the day at 6.00 p.m. feeding mosquitoes on human
larvicidal, pupicidal, smoke repellency effect of arm for experimental purposes was suggested
neem products against malarial vector, (Judson 1967 and 53Briegel 1990). Both females
Anopheles stephensi. and males were provided with 10% glucose
METHODS solution as described (Villani et al., 1983) on
cotton wicks. The cotton was always kept moist
1. Collection of eggs with the solution and changed every day. An egg
The eggs of Anopheles stephensi were collected trap (cup) lined with filter paper containing pure
from local (in and around Coimbatore, India) water was always placed at a corner of the cage.
different breeding habitats with the help of a ‘O’ This arranged made collection of eggs easier.
type brush. The eggs were then brought to the
5. Colonization of mosquito species
laboratory and transferred to 18 x 13 x 4 cm size
enamel trays containing 500 ml water and kept Three species of mosquito as malarial vector,
for larval hatching. They were hatched and Anopheles stephensi were collected and
reared have been still maintained from many maintained at the laboratory by standard
generations in the laboratory. The eggs and procedures. The maintenance of the larvae,
larvae obtained from this stock were used for pupae and adult has done at the standard
different experiments. procedures at the laboratory. Collection and
culture of brackish water mosquitoes were
2. Maintenance of larvae
collected and maintained at the laboratory for
The larvae reared in plastic cups. They were further laboratory and field bioassay.
daily provided with commercial fish food
6. Neem Limonoids
(Lyimo et al., 1992). Water was changed
alternate days. The breeding medium was The neem oil which was procured from the Local
regularly checked and dead larvae were removed Neem Oil Mill, Kalverrampalayam, Coimbatore-
at sight. The normal cultures as well as breeding 641 046, India. Six limonoids (purity 99%), Six

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Murugan et al. Int. J. Pure Appl. Zool., 2(2): 71-83, 2014

neem limonoids (purity>99%), namely control arms were interchanged regularly to

azadiractin, salannin, deacetylgedunin, gedunin, eliminate bias. Each test concentration was
17-hydroxyazadiradione and deacetylnimbin repeated five times and in each replicate subject
were sent from Central Research Laboratories, different volunteers to nullify any effect of
Taiyo Kagaku Co Ltd., Japan. They were colour of the skin on repellent. Volunteers were
dissolved in isopropanol and different
asked to follow the testing protocol.
concentrations were prepared by dilution with
isopropanol. Volunteers conducted their test of each
7. Larvicidal Activity concentration by inserting the treated and control
arms alternatively into a same cage for one full
Laboratory colonies of mosquito larvae were minute for every five minutes. If they were not
used for the larvicidal activity. Twenty-five bitten within 20 minutes, then the arms were
numbers of fourth instar larvae were introduced reinserted for 1 full minute for every 15 minutes,
into the 500 ml glass beaker containing 249 ml of until the first bite occurred.
de-chlorinated water and 1 ml of desired
9. Smoke Toxicity Test
concentrations of neem products was added
separately. Larval food was given for the test Cassia occidentalis parts were used for smoke
larvae. At each tested concentration 3 trials were toxicity assay. The mosquito coil was prepared
made and each trial consisted of five replicates. by following method of Saini et al. (1986) with
Mixing 1ml of acetone with 249 ml of de- minor modifications by using 4 grams from
chlorinated water set up the control. In the plant powdered plant sample considered as active
extracts the larvae exposed to de-chlorinated ingredient and two grams of saw-dust as binding
water without acetone served as control. The material and two grams of coconut shell charcoal
control mortalities were corrected by using powder as burning material. All the three were
Abbott's formula (Abbott's, 1925). thoroughly mixed with distilled water to form a
semisolid paste. Mosquito coils (0.6 cm
Number of dead pupae thickness) were prepared manually from the
Percentage mortality = × 100
Number of pupae introduced semisolid paste and were shade dried. The
LC50, LC90, regression equation and 95% control coils were prepared without plant
confidence limit of lower confidence of limit ingredient.
(LCL) and Upper Confidence Limit (UCL) were The experiments were conducted in glass
calculated from toxicity data by using probit chamber measuring 140 x 120 x 60 cm. A
analysis (Finney, 1971). window measuring 60 x 30 cm was situated at
8. Repellency tests mid bottom of one side of the chamber. Three or
four day’s old blood starved hundred adult
The neem products were evaluated for their female mosquitoes, fed with sucrose solution,
repellent activities against An. stephensi using were released into the chamber. A belly shaven
the human – bait technique (Fradin and Day, pigeon was kept tied inside the case in
2002). First, 1 ml neem oil was mixed with 5 ml immobilized condition. The experimental
coconut oil and diluted in ethanol and prepared 2, chamber was tightly closed. The experiment was
4 and 6% concentrations. For the test, 20 disease repeated five times on five separate days
free, laboratory-reared female mosquitoes were including control using mosquitoes of same age
placed into separate laboratory cages (45 x 38 x groups. The data were pooled and average values
38 cm). Before the test, the volunteer’s skin was were subsequently used for calculations. Control
washed with unscented soap and the tested was maintained in two sets. One set was run with
essential oil was applied from the elbow to the coil lacking the active ingredient of plant powder
finger tips. In each cage one arm was inserted for (control 1) another one was Mortein coil which
one test concentration and the other arm applied was used positive control to compare the
with ethanol served as control. The treated and effectiveness of plant coils.

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Murugan et al. Int. J. Pure Appl. Zool., 2(2): 71-83, 2014

After the experiment was over the fed, unfed Gedunin and Dacetyl nimbin the LC50 and LC90
(active and dead) mosquitoes were counted. The values were increased when compare to
protection given by the smoke from plant Azadiractin. The regression equation of neem oil
samples against the biting of Anopheles stephensi for 4th instar were X= 1.560, Y= -0.784,
was calculated in terms of percentage of unfed azadirachtin were X= 1.682, Y= -0.502, 17-
mosquitoes due to treatment. Hydroxyazadiradione were X= 1.100, Y= -
Number of unfed mosquitoesin treatment - Number of unfed mosquito in control1 0.426, Salannin were X= 1.305, Y= - 0.571,
= × 100
Number of mosquitoes treated Deacetyl gedunin were X= 1.387, Y= - 0.639,
The live blood fed mosquitoes were reared in Dacetyl nimbin were X= 1.353, Y= - 0.804 and
a mosquito cage, measuring 30 x 30 x 15 cm. Gedunin were X= 1.269, Y= - 0.639,
The top and bottom of the cage were fit with respectively.
glass and all other sides were covered with The repellent activity of neem products were
muslin cloth. Water soaked raisins and a 5% tested at three different concentrations (0.2, 0.4,
sucrose solution soaked in cotton balls were and 0.6) of these the neem product of neem oil,
provided as a food source. Water containing Azadirachtin, Salannin, 17-Hydroxy-
powdered yeast and dog biscuits were also kept azadiradione, Deacetyl gedunin, Dacetyl nimbin
inside the cage in a glass bowl for oviposition. and Gedunin exhibited relatively. The neem oil
The eggs from the cage were collected daily till complete protection time at 0.2% concentration,
all the mosquitoes died. A total 50- 100 eggs the production time was 165 mts, at 0.4% the
were allowed to hatch in plastic trays measuring protection time was 248 mts, at 0.6% the
30 x 25 x 6 cm, containing about 2.5 liters of production time was 356 mts. Similarly,
unchlorinated tap water. Hatched larvae’s were Azadirachtin at 0.2% was 134 mts, at 0.4% was
fed with a mixture of dog biscuits and yeast 237 mts, at 0.6% was 348 mts, Salannin at 0.2%
powder in the ratio of 2:1 and water in the tray was 123mts, at 0.4% was 220mts, at 0.6% was
was changed daily. Survival and dead instars 313mts, 17- Deacetyl gedunin at 0.2% was
were counted and reduction in the population 119mts, at 0.4 %was 210 mts, at 0.6% was
from the smoke treated mosquitoes was 304mts, Dacetyl nimbin at 0.2% was 105 mts, at
calculated using the formula. 0.4% was 198 mts, at 0.6% was 289 mts,
Population reduction (%) =
Number of larvae hatched in control - Number of larvae hatched in treated
Number of larvae hatched in control
× 100 Gedunin at 0.2% was 95 mts, at 0.4% was 152
mts, at 0.6% was 206 mts, respectively. The
10. Statistical Analysis neem oil which showed high repellent effect
The data gets from the bioassay subject to (<300 minutes at 0.6 concentration), followed by
statistical analysis. The SPSS software package Gedunin which showed less (<200 minutes at 0.6
was computing all the data including probit concentration). However, the ethanol applied arm
analysis, correlation co-efficient and mean of the served as control provided maximum 7.0 minutes
sample. repellency in this study. The leaves and pods
were also tested smoke repellency bioassay.
RESULTS Smoke emerged from the leaves and pods of had
The neem products also had significant larvicidal potential knock down effect and percentage of
activity. The larval mortality was dose repellency on leaf 59%, pod 53% and with
dependant. The LC50 and LC90 values of commercial coil as positive control which
Azadirachtin treatment against Anopheles showed 65% repellency, respectively. Table 4
stephensi at 0.50, 1.0 and 1.5 ppm concentration shows the result of smoke toxicity effect of
was 0.299% and 1.061%, respectively. After different parts of (leaves and pods) Neem
treatment of neem oil at 0.50, 1.0 and 1.5 ppm ensured population of A. stephensi. The numbers
concentration was 0.503 and 1.324, respectively. of eggs laid by the alive, fed females were
Similarly, Salannin was 0.438% and 1.420%, 17- shown. Number of eggs laid and the hatchability
Hydroxyazadiradione was 0.387% and 1.553%, were greatly reduced or affected by the exposure
Gedunin was 0.558% and 1.568%, of smoke from Neem. The percentage reduction
Deacetylgedunin was 0.461% and 1.384, of hatchability by the smoke from leaves showed
Deacetylnimbin was 0.594% and 1.542%, 57.4 % and from the pods showed 59.2%. The
respectively.After the treatment of salanin, 17- leaves showed a significant effect on the
Hydroxyazadiradione, Deacetyl gedunin, fecundity and hatchability.
76
Table 1. Larvicidal activity of neem products against 4th instar larva of malarial vector, Anopheles.
stephensi.

LC50 (Fiducial LC90 (Fiducial

Mortality (%) Regression Chi square
Neem products limits) limits)
(Mean ± SE) equation (χ2)
(µg/cm2) (µg/cm2)
62.6 ± 0.5 X= 1.682 0.632 0.299 1.061
Azadirachtin 89.82 ± 0.3 Y= -0.502 (0.036) (0.452) (0.940) (1.245)
97.4 ± 0.5
56.2 ± 0.7 X= 1.100 0.479 0.387 1.553
17- 72.6 ± 0.9 Y= - 0.426 (0.034) (0.576) (1.339) (1.967)
Hydroxyazadiradione 90.0 ±0.7
53.6 ± 1.1 X= 1.305 0.438 1.420
76.2 ± 0.9 Y= - 0.571 0.039 (0.177) (0.594) (1.249) (1.714)
Salannin
92.6 ± 0.5
52.0 ± 0.7 X= 1.387 0.100 0.461 1.384
77.6 ± 0.9 Y= - 0.639 (0.228) (0.606) (1.226) (1.649)
Deacetyl gedunin
92.4 ± 0.9
51.4 ± 1.0 X= 1.560 0.843 0.503 1.324
75.2 ± 0.8 Y= -0.784 (0.314) (0.628) (1.183) (2.548)
Neem oil
95.2 ± 0.8
48.0 ± 0.7 X= 1.269 0.558 1.568
69.4 ± 0.6 Y= - 0.639 0.265 (0.339) (0.699) (1.377) (1.903)
Gedunin
89.2 ± 0.8
46.4 ± 1.2 X= 1.353 0.632 0.594 1.542
Dacetyl nimbin
68.0 ±1.2 Y= - 0.804 (0.403) (0.723) (1.364) (1.844)
90.2 ± 0.5

Table 2.Repellent activity of neem products against malarial vector, An. stephensi.
Concentration Complete – protection time (min)
Neem products
(%) Treated Control
0.2 165.0 ±1.0a 6.0 ± 1.0
Neem oil 0.4 248.0 ± 1.1a 4.0 ± 1.0
0.6 356.0 ± 1.4a 5.0 ± 1.0
0.2 134.0 ± 1.3b 5.0 ±1.0
Azadirachtin 0.4 237.0 ± 1.2b 7.0 ±1.0
0.6 348.0 ± 0.7a 4.0 ±1.0
0.2 123.0 ± 1.0c 5.0 ±1.0
Salannin 0.4 220.0 ± 0.7b 6.0 ±1.0
0.6 313.0 ± 0.4a 5.0 ±1.0
0.2 119 ± 0.7a 5.0 ±1.0
Deacetylgedunin 0.4 210 ± 0.7a 6.0 ±1.0
0.6 304 ± 1.0b 5.0 ±1.0
0.2 109 ± 0.4a 6.0 ±1.0
17-Hydroxyazadiradione 0.4 201 ± 0.3a 4.0 ±1.0
0.6 307 ± 0.4a 5.0 ±1.0
0.2 105 ± 0.7a 5.0 ±1.0
Deacetylnimbin 0.4 198 ± 1.3b 5.0 ±1.0
0.6 289 ± 0.7a 6.0 ±1.0
0.2 95.0 ± 1.1b 4.0 ± 1.0
Gedunin 0.4 152.0 ± 1.0b 4.0 ± 1.0
0.6 206.0 ± 0.7a 6.0 ± 1.0
Each value (mean ± SE) represents mean of five values. Within a column means followed by the same
letter (s) are not significantly different at 5% level by DMRT.
Control I *: ethanol.

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Murugan et al. Int. J. Pure Appl. Zool., 2(2): 71-83, 2014

Table 3. Smoke toxicity effect of leaves of neem against biting activity of Anopheles stephensi.
Neem No. of Fed Unfed Total % unfed
parts used in mosquito mosquito mosquito over control
grams tested Alive Dead I
Leaf 2 G 100 16b 36 48 84 59
Pods 2 G 100 22c 42 36 78 53
Control I * 100 75 25 0 25 0
Control II * 100 10a 47 43 90 65

Within a column means followed by the same letter (s) are not significantly different at 5% level by
DMRT.
Control I *: Negative control – blank without plant material.
material
Control II *: Positive control – mortein coil.

Table 4. Smoke toxicity effect of leaves of Azadirachta indica on reproduction and survival of
Anopheles stephensi.
Azadirachta No. of Total No. of Total No. of larva % of reduction in
indica mosquito eggs hatched from the population over
parts used tested eggs control I
Leaf 25 1045 534 57.4
Pod 25 950 512 59.2
Control I * 25 1450 1256 0
Control II * 25 820 386 69.2
Control I * : Negative control – blank without plant material.
material
Control II *: Positive control – mortein coil.
coil

Figure 1. Structure of neem limonoid.

78
DISCUSSION quinquefasciatus to ether extract of C. inerme
leaves resulted in death at larval–pupal molt and
Malaria is the largest single component of
pupal–adult eclosion and suggesting inhibition of
disease burden; epidemic malaria, in particular,
the moulting process (Pereira and Gurudutt,
remains a major public health concern in tropical
1990) lend further support to our observations.
countries. In many developing countries, and
especially in Africa, malaria exacts an enormous Lavie et al. (1967) reported that isolated
toll in lives, in medical costs, and in days of gedunin and 7-deacetyl gedunin from neem seed
labor lost (Lambert, 2005). Since from the oil and bark and 7-benzoyl derivative from dried
ancient years the plants were being used for the seeds. Gedunin was shown to posses both
control of mosquitoes in many ways like burning antifungal and antimalarial properties. The
the plant parts, keeping the plants near window antifungal and antimalarial properties may be
etc., In the present study also we have shown that effected to the malarial vector, Anopheles
neem product has larvicidal as well as repellent stephensi. Dhar et al. (1996) demonstrated that
activity against various species of mosquitoes. the inhibitory effect of neem oil volatiles on
Recently, strong repellent actions of gonotropic cycle in An. stephensi and An.
Azadirachata indica, Cymbopogan martinii var. culicifacies. A neem oil formulation containing
sofia, C.citratus, C.naridus, Ocimum spp etc. 32% neem seed oil (an equivalent of 0.03%
have been reported against An. culicifacies and azadirachtin), an emulsifier (5%) and 63% iso
other species of mosquitoes (Ansari and Razdan, propanol (solvent) was investigated for its
1994, 1995; Sharma et al., 1993; Sharma and larvicidal activities against An. gambiae (Okumu,
Ansari, 1994). These findings have re- et al., 2007). It was toxic to mosquito larvae
emphasized the need to explore the possibility of with LC50 value of 11 ppm and also reported to
using herbal-based repellents (Osmani et al., possess insect growth regulators. Gianotti and co
1972) as supplementary and complimentary workers (Gianotti, et al., 2008) used powdered
measures for malaria control. This will reduce seeds of neem trees and applied twice a week to
the chemical burden on the environment. The known breeding sites for An. gambiae at the rate
neem oil more potential effect against mosquito of 10 gm/m2 of pool surface area for effective
larvae. Murugan et al. (1999) (established the larval control. Azadirachtin acts as anti-
neem seed kernel extract possess anti-pupational ecdysteroid and kills larvae by growth inhibition
property for mosquito species. Babu and effect (Zebit, 1984). In the present investigation,
Murugan (1996) investigated that the larvicidal neem products was found effective to control
effect of resinous exudate from the tender leaves mosquito larvae in laboratory conditions and
of Azadirachta indica. Vahitha et al. (2002) more than 90% reduction of Anopheles stephensi
studied the larvicidal efficacy of Pavonia larvae were observed up to three weeks of post
zeylamica L. and Acacia ferruginea D.C. against application. Limonoid compounds are found in
Culex quinquefasciatus Say. The present study almost all parts of the neem plant. The active
showed that, after the treatment of salanin, 17- ingredients in Neem extracts are very low in
Hydroxyazadiradione, Deacetyl gedunin, toxicity and thus are not toxic to mammals and
Gedunin and Dacetyl nimbin, the LC50 and LC90 also quickly biodegrade from the sun’s light.
values were increased when compare to However, the use of Azadirachtin based neem
Azadirachtin. The most active constituent, pesticides may be limited by the acid and base
azadirachtin, a triterpenoid, has show to have sensitivity of the compound and its susceptibility
properties including feeding and ovipositional to photo degradation due to presence of light-
deterrence, repellency, growth disrurption, absorbing moieties (Stokes and Redfern, 1982;
reduced fitness and sterility in a number of Barnby et al., 1989). Hence, the neem oil not
species of hemimetabolous and holometabolous effected to human skin and not makes allergic
insects (Ascher and Meisner, 1989; Schmutterer, reaction to human. The neem oil affected to
1990). From the light of literature we came to target organism malarial vector, Anopheles
know about many larvicidal studies conducted stephensi only. Subramonia Thangam and
using plants and results clearly suggested that the Karthiresan (1992) stated that smoke from
C. inerme interfered with developmental burning various dry materials has been used
processes of the fourth instar larvae and pupae of since early times to deter insects, especially
A. aegypti. In this context, the observations that mosquitoes and studied smoke repellency effect
exposure of fourth instar mosquito Culex of marine plants against Culex quinquefasciatus.

*Corresponding author e-mail: kmvvkg@gmail.com, Mobile: +91 9894832849

Murugan et al. Int. J. Pure Appl. Zool., 2(2): 71-83, 2014

(Schmutterer (1990) stated that when an insect animals. In: The Neem Tree (Ed. M.
eats azadirachtin, it actively attacks the insect’s Jacobsen), boca raton (CRS Press), p. 113.
reproductive cycle, its feeding pattern, its bodily
Babu, R. and Murugan, K., 1998. Interactive
development, as well as acting direct toxin. Thus,
effect of neem seed kernel extract and neem
when azadirachtin is sprayed on the plant and the
gum extract on the control of culex
insect take a bite – if it can stand the bitter taste –
quinquefasciatus say. Neem Newslett., 15
the insect will no longer be able to reproduce, eat
(2): 9-11.
and grow. Similarly, The adverse effect of smoke
exposure on adult mosquito which prevents egg Babu, R. and Murugan, K., 2000. Larvicidal
laying and hatching may be due to interference effect of resinous exudates from the tender
of endocrine events causing reproductive leaves of Azadirachta indica. Neem
malfunction. Newsletter., 17:1.
CONCLUSION Banchio, E., Valladares, G., Defago, M.,
From this study it has been concluded that based Palacios, S. and Carpinella, C., 2003. Effects
on the larvicidal, smoke repellent properties of Melia azedarach (Meliaceae) fruit extracts
neem products showed mosquitocidal properties. on the leafminer Liriomyza huidobrensis
Hence, it can be concluded that neem as effective (Diptera: Agromyzidae): assessment in
biopesticides for the Integrated Vector Control laboratory and field experiments. Ann. Appl.
Program. Moreover, neem is indigenous, Biol., 143: 187-193.
medicinal importance and much of social Barnby, M.A., Yamasaki, R.B. and Klocke, J.A.,
relevance to people and traditional knowledge of 1989. J. Econ. Entomol., 82: 58-62.
our country.
Briegel, H., 1990. Fecundity, metabolism and
CONFLICTS OF INTEREST body size in Anopheles stephensi (Diptera:
The authors declare that there are no conflicts of Culicidae) vector of malaria. J. Med.
interest associated with this article. Entomol., 27: 839-856.

ACKNOWLEDGEMENTS Champagne, D.E., Koul, O., Isman, M.B.,

Scudder, G.G.E. and Towers, G.H.N., 1992.
The authors are grateful to Professor and Head, Phytochemistry., 31: 377-94.
Department of Zoology, Bharathiar University
Chatterjee, A. and Pakrashi, S., 1994. The
for the laboratory facilities providing for this
experiment. Treatise on Indian Medicinal Plants., Vol. 3,
p. 76.
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