Malaria Journal BioMed Central

Research Open Access

Larvicidal activity of neem oil (Azadirachta indica) formulation
against mosquitoes
Virendra K Dua*1, Akhilesh C Pandey1, Kamaraju Raghavendra2,
Ashish Gupta1, Trilochan Sharma1 and Aditya P Dash2

Address: 1National Institute of Malaria Research, Field Unit, Sector-III, BHEL, Hardwar 249043, India and 2National Institute of Malaria Research,
22 Sham Nath Marg, Delhi 110054, India
Email: Virendra K Dua* - vkdua51@gmail.com; Akhilesh C Pandey - acpandey62@gmail.com;
Kamaraju Raghavendra - kamarajur2000@yahoo.com; Ashish Gupta - gupta4268@gmail.com; Trilochan Sharma - trilochan79@gmail.com;
Aditya P Dash - apdash2@rediffmail.com
* Corresponding author

Published: 8 June 2009 Received: 8 March 2009

Accepted: 8 June 2009
Malaria Journal 2009, 8:124 doi:10.1186/1475-2875-8-124
This article is available from: http://www.malariajournal.com/content/8/1/124
© 2009 Dua et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Mosquitoes transmit serious human diseases, causing millions of deaths every year. Use of synthetic
insecticides to control vector mosquitoes has caused physiological resistance and adverse environmental effects in
addition to high operational cost. Insecticides of botanical origin have been reported as useful for control of mosquitoes.
Azadirachta indica (Meliaceae) and its derived products have shown a variety of insecticidal properties. The present paper
discusses the larvicidal activity of neem-based biopesticide for the control of mosquitoes.
Methods: Larvicidal efficacy of an emulsified concentrate of neem oil formulation (neem oil with polyoxyethylene ether,
sorbitan dioleate and epichlorohydrin) developed by BMR & Company, Pune, India, was evaluated against late 3rd and
early 4th instar larvae of different genera of mosquitoes. The larvae were exposed to different concentrations (0.5–5.0
ppm) of the formulation along with untreated control. Larvicidal activity of the formulation was also evaluated in field
against Anopheles, Culex, and Aedes mosquitoes. The formulation was diluted with equal volumes of water and applied @
140 mg a.i./m2 to different mosquito breeding sites with the help of pre calibrated knapsack sprayer. Larval density was
determined at pre and post application of the formulation using a standard dipper.
Results: Median lethal concentration (LC50) of the formulation against Anopheles stephensi, Culex quinquefasciatus and
Aedes aegypti was found to be 1.6, 1.8 and 1.7 ppm respectively. LC50 values of the formulation stored at 26°C, 40°C and
45°C for 48 hours against Ae. aegypti were 1.7, 1.7, 1.8 ppm while LC90 values were 3.7, 3.7 and 3.8 ppm respectively.
Further no significant difference in LC50 and LC90 values of the formulation was observed against Ae. aegypti during 18
months storage period at room temperature. An application of the formulation at the rate of 140 mg a.i./m2 in different
breeding sites under natural field conditions provided 98.1% reduction of Anopheles larvae on day 1; thereafter 100%
reduction was recorded up to week 1 and more than 80% reduction up to week 3, while percent reduction against Culex
larvae was 95.5% on day 1, and thereafter 80% reduction was achieved up to week 3. The formulation also showed 95.1%
and, 99.7% reduction of Aedes larvae on day 1 and day 2 respectively; thereafter 100% larval control was observed up to
day 7.
Conclusion: The neem oil formulation was found effective in controlling mosquito larvae in different breeding sites
under natural field conditions. As neem trees are widely distributed in India, their formulations may prove to be an
effective and eco-friendly larvicide, which could be used as an alternative for malaria control.

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Background primary vector of urban malaria,Culex quinquefasciatus a

Mosquitoes transmit serious human diseases like malaria, common vector of filariasis, and Aedes aegypti a common
filariasis, Japanese encephalitis, dengue haemorrhagic vector of dengue, dengue haemorrhagic fever and yellow
fever and yellow fever causing millions of deaths every fever. The larvae were obtained from laboratory-estab-
year [1]. Extensive use of chemical insecticides for control lished colony as described earlier [12]. Twenty-five larvae
of vector borne diseases has created problems related to were released into 500 ml glass beakers containing 250 ml
physiological resistance to vectors, adverse environmental distilled water. The larvae were provided a mixture of dog
effects, high operational cost and community acceptance biscuit and yeast powder in a 3:2 ratio as nutrients and
[2]. Numerous plant products have been reported either supplemented with different concentrations (0.5 to 5.0
as insecticides for killing larvae or adult mosquitoes or as ppm) of the formulation. The experiments were carried
repellents for mosquito biting and are one of the best out at 26°C ± 2°C. Five replicates of each concentration
alternatives for mosquito control [2,3]. were run under the same microclimatic conditions along
with untreated control. Mortality of larvae was monitored
Neem trees, (Azadirachta indica) native of India, belonging at 24 hours. The percent corrected mortality was calcu-
to family Meliaceae are fast growing evergreen trees rang- lated using Abbott's formula [13] and Log probit analysis
ing in height from 12 – 24 m. They are widespread in trop- was used to determine the median lethal concentration
ical and subtropical regions of the world, including semi- (LC50)/90% lethal concentration (LC90) of the formula-
arid and wet- tropical regions [4]. Neem seeds contain tion.
approximately 99 biologically active compounds of
which azadirachtin, nimbin, nimbidin and nimbolides Stability test
are major molecules. Many of these derived products have Larvicidal bioassay of the neem oil-based formulation
antifeedancy, ovicidal activity, fecundity suppression stored at 26°C, 40°C and 45°C for 48 hrs was evaluated
besides insect growth regulation and repellency against against Ae. aegypti at different concentration (0.5–5.0
insects [5-10]. Neem products have low toxicity to birds, ppm) as per the method reported above. Five replicates of
fish and mammals and are less likely to induce resistance each concentration were run under the same microcli-
due to their multiple mode of action on insects. In addi- matic conditions along with untreated control. A total of
tion to this, insect growth regulatory activity of neem 100 larvae of Ae. aegypti were exposed against each con-
weakens the cuticle defence system of the larvae causing centration. Further bioassay test of the neem oil formula-
easy penetration of pathogenic organisms into insect sys- tion of different concentrations (0.5–5.0 ppm) stored at
tem. Azadirachtin, a biologically active compound has room temperature (26°C ± 2°C) was evaluated against
been promoted as a new insecticide that is considered Ae. aegypti larvae at three months interval period for 18
more eco- friendly than synthetic insecticides. The pesti- months. Three replicates of each concentration were car-
cidal efficacy, environmental safety and public acceptabil- ried out along with control.
ity of neem and its products for control of crop pests has
led to its adoption into various mosquito control pro- Field evaluation of larvicidal activity
grammes [8,11]. Field evaluation of larvicidal activity of the neem oil for-
mulation was carried out in two districts of Uttar Pradesh
The present study was aimed to determine the larvicidal viz. Mathura and Kanpur and district Hardwar of the state
potential of the emulsified neem oil formulation against of Uttarakhand. Initially a survey was carried out to ascer-
different mosquito genera under natural field conditions tain the suitability of different breeding habitats of target
in India. species for the trial. Larvicidal efficacy was carried out
against late 3rd and early 4th instar larvae of different gen-
Methods era of mosquitoes under natural field conditions.
Neem oil formulation
The test formulation was an emulsified concentrate con- Larval density was determined using standard dipper (300
taining 0.15% w/v azadirachtin, polyoxyethylene ether ml capacity with 9 cm diameter) method. Treatment dose
(emulsifier), sorbitan dioleate (surfactant) and epichloro- (140 mg a.i./m2) of the formulation was determined on
hydrin (used as a stabiliser to protect the degradation of the basis of the results of laboratory evaluation carried out
the formulation under exposure to sun light.), developed against Cx. quinquefasciatus and Ae. aegypti [14]. Five liters
by BMR & Company, Pune, India was evaluated against of the neem oil formulation was mixed with equal volume
late 3rd and early 4th instar larvae of different genera of of water to make a uniform suspension and applied to 53
mosquitoes. m2 surface area of breeding habitats through precalibrated
knapsack sprayer.
Larvicidal bioassay
Larvicidal bioassay of the formulation was performed on Larval density/dip was recorded a day before application
late 3rd and early 4th instar larvae of Anopheles stephensi, a for both experimental and control habitats, thereafter

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observations were recorded at 24, 48 and 72 hr of post Table 2: Larvicidal activity of neem oil formulation against Aedes
application. Further observations were made at weekly aegypti at different storage temperature
intervals for 3 weeks. Percent reduction was calculated for Storage temperature Larvicidal activity (mean)
3rd & 4th instar larvae and pupae using the formula.
LC50 LC90
C ×T
%Reduction = 100 − I 2 × 100 26 ± 2°C 1.7 ± 0.5 3.7 ± 0.8
C 2 × T1

C1, C2 are pre-treatment, post-treatment larval density in 40°C 1.7 ± 0.6 3.7 ± 0.5
control whereas T1, T2 are pre-treatment post-treatment,
immature density in experimental habitats respectively. 45°C 1.8 ± 0.4 3.8 ± 0.6

Number of each replicate: 3

Results
Laboratory study
Mean LC50 and LC90 values (95% confidence limits) of the tanks, pits, drains were also carried at Indian Oil Corpora-
neem oil formulation against An. stephensi, Cx. quinquefas- tion, Mathura and Bharat Heavy Electricals Limited, Hard-
ciatus and Ae, aegypti are given in Table 1. Mean LC50 val- war for Culex and Anopheles breeding. Culex and Anopheles
ues of the formulation were 1.6, 1.8 and 1.7 ppm while larvae collected from these sites were identified as Cx.
LC90 were 3.4, 3.5 and 3.7 ppm against An. stephensi, Cx. quinquefasciatus, An. culicifacies and An. subpictus respec-
quinquefasciatus and Ae. aegypti respectively. Results of sta- tively.
bility test of the neem oil based formulation stored at dif-
ferent temperatures against Ae. aegypti are given in Table 2. Mean percent reduction of larval density against Cx. quin-
LC50 values of the biopesticide stored at 26°C, 40°C and quefasciatus and anophelines in different breeding habi-
45°C for 48 hours were 1.7, 1.7, 1.8 ppm while LC90 val- tats are given in Table 3. In pits, percent reduction of Culex
ues recorded were 3.7, 3.7 and 3.8 ppm respectively. Mean larvae was 95.9, 90.2, 87.2 on days 1, 2, 3 respectively of
LC50 and LC90 of the formulation during 18 months stor- post application, while more than 70% reduction was
age period at room temperature against Aedes aegypti is observed up to week 3. In tanks 91.6% – 92.4% reduction
shown in Figure 1. No significant difference (P > 0.5) in of Culex larvae was observed up to day 7 of post applica-
LC50 and LC90 value of the formulation was observed. tion thereafter 80.7% reduction was noted up to week 3,
while in drains there was more than 90% larval control up
Field study to day 7 and remained above 75% up to week 3. An effec-
Before start of the study, a preliminary survey of various tive control of late instars anopheline larvae in tanks was
breeding sites of mosquitoes was carried out. In all 73 observed with 98.2% reduction on day 1, followed by
breeding sites (factory scraps) were surveyed inside the 100% reduction up to day 7 and more than 75% reduc-
Ordnance factory, Kanpur, out of which 67 sites were tion till week 3. In pits, 96.2% control was recorded on
found positive (91.8%) for Aedes larvae. Larvae collected day 1, 100% up to day 7 and more than 75% reduction up
from these breeding sites were identified as Ae. aegypti and to week 3. The mean percent reduction of Culex larvae was
Ae. albopictus. A survey of different breeding sites such as 89.9–95.5% up to day 7 followed by 79.7–85.7% up to
week 3, while for Anopheles larvae mean percent reduction
Table 1: Larvicidal activity of neem oil formulation against
was 90.4–100% up to day 7 followed by 83.8–90.4% up
mosquito in laboratory to week 3 (Figure 2).

Species Larvicidal activity (ppm) Larvicidal activity of the formulation against Aedes larvae
in different breeding sites is given in Table 4. There was
LC50 LC90
(Mean ± sd) (Mean ± sd)
85.2% to 98.1% reduction of Aedes larvae on day 1 of post
application of the neem oil formulation, thereafter 99.7%
Anopheles stephensi 1.6 ± 0.4 3.4 ± 0.5 to 100% reduction was recorded up to day 7.
(1.1 – 2.5)* (2.7 – 4.0)
Discussion
Culex quinquefasciatus 1.8 ± 0.5 3.5 ± 0.6 Neem trees are found throughout India with a myriad of
(1.2 – 2.6) (2.8 – 4.2) uses in medicine, as well as pest control [4]. Neem-based
pesticides are now extensively used in agriculture practices
Aedes aegypti 1.7 ± 0.3 3.7 ± 0.5
all over the world. It contains azadirachtin, which is a pre-
(1.3 – 2.1) (3.1 – 4.3)
dominant insecticidal active ingredient, having antefeed-
Water depth: 2.5 cm; * 95% confidence limits; Number of replicates: 5 ent, ovipositional deterrence repellency, growth

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Stability
Figure 1test of the neem oil formulation against Aedes aegypti
Stability test of the neem oil formulation against Aedes aegypti.

disruption, sterility and larvicidal action against insects an application of neem cake powder resulted in drastic
[6]. There are various reports of control of mosquito reduction in the late instar larvae and pupae of culicine
breeding under field conditions. An emulsion of neem oil mosquitoes in paddy field [16].
in water was found to be effective in controlling breeding
of Cx. quinquefasciatus, An. stephensi and Ae. aegypti in Mean LC50 values were 1.6, 1.8 and 1.7 ppm against An.
pools, tanks and coolers up to 2 to 3 weeks [15], whereas stephensi, Cx. quinquefasciatus and Ae. aegypti, while LC90
Table 3: Larvicidal activity of neem oil formulation against mosquitoes larvae in field

Mosquito species Breeding sites Pre treatment Percent reduction of larval density (mean ± sd)
density

Day-1 Day-2 Day-3 Week-1 Week-2 Week-3

Culex Pits 28.9 ± 10.6 95.9 ± 3.5 90.2 ± 6.9 87.2 ± 11.0 87.5 ± 8.2 85.9 ± 8.0 80.5 ± 7.3

Tanks 26.8 ± 11.5 91.9 ± 5.8 93.2 ± 3.2 97.7 ± 1.9 92.4 ± 8.0 86.2 ± 8.2 80.7 ± 9.2

Drains 115.7 ± 64.6 99.4 ± 0.6 98.8 ± 1.2 98.6 ± 1.4 84.9 ± 4.6 85.0 ± 11.8 77.8 ± 11.0

95.5 ± 4.1 94.1 ± 4.3 94.5 ± 5.5 89.9 ± 2.5 85.7 ± 0.8 79.7 ± 1.6

Anopheles Pits 13.5 ± 7.5 96.2 ± 4.5 100 100 100 85.4 ± 14.1 76.6 ± 9.6

Tanks 10.4 ± 5.7 98.2 ± 1.8 100 100 100 87.0 ± 9.7 77.7 ± 10.0

Drains 13.0 ± 6.7 100 100 100 100 98.7 ± 1.3 97.0 ± 3.0

98.1 ± 1.9 100 100 100 90.4 ± 7.2 83.8 ± 11.5

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100% reduction was observed in Anopheles larvae up to

week 1, after that more than 80% reduction was recorded
up to week 3.

Dhar et al [17] demonstrated the inhibitory effect of neem

oil volatiles on gonotropic cycle in An. stephensi and An.
culicifacies. A neem oil formulation containing 32% neem
seed oil (an equivalent of 0.03% azadirachtin), an emul-
sifier (5%) and 63% iso propanol (solvent) was investi-
gated for its larvicidal activities against An. gambiae [18]. It
was toxic to mosquito larvae with LC50 value of 11 ppm
and also reported to possess insect growth regulators.
Gianotti and co workers [19] used powdered seeds of
neem trees and applied twice a week to known breeding
sites for An. gambiae at the rate of 10 gm/m2 of pool sur-
face area for effective larval control. Azadirachtin acts as
Figureof
Impact
against mosquito
2 larvicidallarvae
activity of the neem oil formulation anti-ecdysteroid and kills larvae by growth inhibition
Impact of larvicidal activity of the neem oil formula- effect [20]. In the present investigation, neem oil formula-
tion against mosquito larvae. tion was found effective to control mosquito larvae in dif-
ferent breeding habitats under natural field conditions
and more than 80% reduction of Anopheles, Culex and
were 3.4, 3.5 and 3.7 ppm respectively. LC50 of the formu- Aedes larvae was observed up to three weeks of post appli-
lation stored at 26°C, 40°C and 45°C for 48 hours were cation.
1.7, 1.7, 1.8 ppm while LC90 values were 3.7, 3.7 and 3.8
ppm respectively which revealed that there was no differ- Neem-based biopesticides and neem extracts have a wide
ence in the biopestcidal activity of the neem oil formula- range of effects against insect pests including repellence,
tion at different storage temperatures. No significant feeding, toxicity, sterility and growth regulator activity
difference of larvicidal activity of the formulation was also and are relatively safe towards non- target biota with only
observed during 18 months storage period at room tem- minimal risk of direct adverse effects on aquatic biota
perature. from contamination of water bodies [21,22]. Allelochem-
icals such as azadirachtin, nimbin, nimbidin, nimbolides,
In the present study an application of the formulation at nimolic acid, salannin, melianttriol, azadirachtol present
the rate of 140 mg a.i./m2 in pits, tanks and drains pro- in neem affect the biochemical and physiological proc-
vided above 90% reduction of Culex larvae up to week 1 esses of insect system and nullify the insect detoxification
and thereafter 80% reduction up to week 3, whereas mechanism thereby not allowing the pest to develop

Table 4: Larvicidal activity of neem oil formulation against Aedes mosquitoes in field

Breeding sites Pre treatment larval density pH Percent reduction of larval density (Mean ± sd)

Day-1 Day-2 Day-3 Day-7

Tyres 10.3 ± 4.1 8.0–9.0 94.3 ± 4.5 98.6 ± 1.4 100 100

Machinery scraps 14.5 ± 8.6 8.0–9.0 96.0 ± 3.0 100 100 100

Iron container 19.2 ± 5.7 8.5 98.1 ± 1.5 100 100 100

Iron box 11.0 ± 6.0 8.0–8.5 96.9 ± 2.0 100 100 100

Iron tanks 9.0 ± 2.6 8.0–8.5 85.2 ± 6.5 100 100 100

Plastic scrap 6.0 8.5 100 100 100 100

95.1 ± 5.2 99.7 ± 0.3 100 100

Total replicates: 21

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Acknowledgements
The investigators are thankful to Director General, Indian Council of Med-
Publish with Bio Med Central and every
ical Research and Director, National Institute of Malaria Research for grant-
scientist can read your work free of charge
ing permission to under take the study. We are also thankful to BMR & Co. "BioMed Central will be the most significant development for
Pune for sponsoring the project and supply of the formulation for the trials. disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UK
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