全 文 ：昆 虫 学 报 Acta Entomologica Sinica，July 2011，54(7):793 － 799 ISSN 0454-6296
* Corresponding author，E-mail:sarita． sanjay90@ gmail． com
Received:2010-08-27;Accepted:2011-03-17
Larvicidal potential of commercially available pine (Pinus
longifolia)and cinnamon (Cinnamomum zeylanicum)oils
against dengue fever mosquito，Aedes aegypti L．
(Diptera:Culicidae)
Radhika WARIKOO，Naim WAHAB，Sarita KUMAR*
(Department of Zoology，Acharya Narendra Dev College，University of Delhi，Kalka Ji，New Delhi 110019，India)
Abstract:The aromatic nature of pine and cinnamon oils has established them as good adult repellents
but their larvicidal efficacy against mosquitoes has not been explored much． Keeping this in view，
laboratory studies were conducted to uncover the larvicidal potential of commercially available pine
(Pinus longifolia)oil and cinnamon (Cinnamomum zeylanicum)oil against the early 4th instar larvae of
Aedes aegypti，originating from Delhi，India． The larvicidal effects were investigated in terms of the larval
mortality，behavioural and morphological changes，if any． Our studies revealed the larvicidal efficiency of
both the oils against 4th instar larvae of Ae． aegypti，the pine oil proving to be more effective than the
cinnamon oil． The LC50 and LC70values exhibited by pine oil were 0． 33093 mg /L and 0． 54476 mg /L，
respectively，while the values obtained with cinnamon oil were 0． 63159 mg /L and 0． 77736 mg /L，
respectively． It was further observed that at LC90 the larvicidal potential of cinnamon oil surpassed that of
the pine oil，exhibiting a value of 1． 11879 mg /L as in comparison to 1． 04915 mg /L shown by pine oil．
The behavioural changes as excitation，restlessness，tremors，and convulsions followed by paralysis
observed in the treated larvae suggest a probable effect of the oils on their neuromuscular system．
Microscopic study of morphological alterations in the treated larvae revealed that most of their organs had
a normal structural appearance as that of controls except the little internal shrinkage in anal gills leading
to the structural deformity． This indicates the anal gills as the probable action sites of the oil extracts and
dysfunction of the gills leading to larval mortality． The potential of oils as new types of larvicides for the
control of mosquitoes are explored．
Key words:Aedes aegypti;pine;cinnamon;larvicidal potential;anal gills;convulsions
1 INTRODUCTION
The vector-borne diseases caused by mosquitoes
are one of the major health problems in many
countries (Kannathasan et al．，2007)． Besides
being a summer nuisance，mosquitoes pose some
major public health problems，carrying pathogens of
the deadly diseases such as dengue， malaria，
encephalitis， yellow fever， chikungunya， and
filariasis． These vectors occur mainly in tropical
countries where more than two billion people live in
endemic regions with about one million deaths been
claimed yearly from malaria and filariasis
(Southgate，1984;WHO，2005)． In the absence of
effective preventive measures or vaccine，the best
approach should be the interruption of disease
transmission by either killing，preventing mosquitoes
from biting people or by killing the larva at the
breeding sites of vectors． The control of mosquitoes
has been complicated because the repeated use of
chemical insecticides has disrupted natural biological
control systems sometimes resulting in the
widespread development of resistance． These
problems have warranted the need for search and
development of alternative strategies using eco-
friendly products which are environmentally safe，
biodegradable and low cost larvicides and adultcides
for killing larval and adult mosquitoes respectively
from natural sources (Mittal and Subbarao，2003)．
Natural products are the best option because of their
less harmful nature to environment and non-targeted
organisms． Plants offer an alternative source of
insect-control agents because they contain a range of
bioactive chemicals，many of which are selective and
have little or no harmful effect on non-target
organisms and the environment (Arnason et al．，
1989;Hedlin et al．，1997)． In recent years，much
effort has，therefore，been focused on plant extracts
or phytochemicals as potential sources of mosquito
control agents or as lead compounds (Sukumar et al．，
DOI:10.16380/j.kcxb.2011.07.007
794 昆虫学报 Acta Entomologica Sinica 54 卷
1991;Ansari et al．，2005;Singh et al．，2006)．
Several extracts and compounds from different plant
families have been evaluated to show new and
promising larvicides (Markouk et al．， 2000;
Prabakar and Jebanesan， 2004; Mohan and
Ramaswamy， 2007; Innocent et al．， 2008a，
2008b) ，however very few plant products have been
developed for controlling mosquitoes． The plant oils
have received much attention as potentially useful
bioactive compounds against insects showing a broad
spectrum of activity against insects，low mammalian
toxicity and degrading rapidly in the environment
(Kim et al．， 2001 )． Keeping in mind the
availability， low budget and less environmental
impact， the experiments were performed on the
commonly available essential oils already in domestic
use to assess their mosquiticidal potential．
Pine oil and cinnamon oil show promise as a
great-smelling，environmentally friendly pesticide，
with the ability to kill mosquito larvae (Cheng et al．，
2004; Ansari et al．， 2005)． Pinus longifolia
(Family: Pinaceae) commonly known as pine，
yields oil which is traditionally used for the
protection from mosquito bites． It is also used as a
herbal medicine in some rural areas in India． In
addition to oil，resins of the pine have been used as
a mounting medium for the preservation of insects
(Ansari et al．，2005)． Cinnamon oil is obtained
from the bark and leaves of cinnamon tree，
Cinnamomum zeylanicum． Cinnamon， due to its
medicinal properties，has been used in many cultures
for treating a variety of health disorders．
Cinnamaldehyde is the main constituent in cinnamon
leaf oil and is used worldwide as a food additive and
flavouring agent，and as mosquito repellent (Cheng
et al．，2004)． Much work has not been carried out
regarding the larvicidal effects of the pine and
cinnamon oil against mosquitoes． Present studies，
thus， were carried out to study the larvicidal
potential of the commercially available pine and
cinnamon oils against dengue fever mosquito，Ae．
aegypti．
2 MATERIALS AND METHODS
2． 1 Mosquito culture
The present investigations employ the third
instar and the early fourth instar larvae of Ae． aegypti
originated from field-collected engorged female adults
from Delhi，India． The colony was maintained in an
insectary without any insecticide exposure at 28 ±
1℃，80% ±5% RH and 14L∶ 10D photoperiod
(Kumar et al．，2002)． Adults were supplied with
soaked deseeded raisins． Periodic blood meals were
provided to female mosquitoes for egg maturation by
keeping restrained albino rats in the cages． The eggs
were collected in an enamel bowl lined with
Whatman filter paper and were allowed to hatch in
enamel trays filled with de-chlorinated water． Larvae
were fed upon a mixture of yeast powder and grinded
dog biscuits． The pupae formed were collected in
enamel bowls and transferred to the cloth cages for
adult emergence．
2． 2 Larvicidal bioassay
The larvicidal bioassay was performed at 28 ±
1℃on the Ae． aegypti larvae in accordance with the
WHO method for mosquito larvae (Kumar et al．，
2002)． The graded series of each oil was prepared
using ethanol as the solvent． The early 4th instar
larvae of Ae． aegypti，in batches of 25，were taken
in plastic bowls containing 99 mL of distilled water
and transferred to glass jar containing 150 mL of
distilled water and 1 mL of the particular
concentration of oil． Four replicates were carried out
simultaneously for each dilution making a total of 100
larvae for each concentration． Controls were exposed
to the solvent，i． e． ethanol alone．
2． 3 Behavioural study
Immediately after treatment，symptoms in treated
larvae were observed and recorded at frequent time
intervals． During the treatment period no food was
offered to the larvae． The larvae were considered
moribund if，at the end of 24 h，they showed no sign
of swimming movements even after gentle touching with
a glass rod． The dead and moribund larvae were
recorded after 24 h as larval mortality．
2． 4 Morphological study
The dead larvae treated with a lethal dosage
(LC99)of each oil，separately，were mounted with
Hoyer’s medium on a microscope slide and scrutinized
for morphological alterations under light microscopy．
Morphological changes in body segments including the
head，thorax，and abdomen，and other organs such as
the eyes，antennae，mouth brushes，setae，saddle，
and anal gills were observed， photographed， and
compared with those of the controls．
2． 5 Data statistics and analysis
The larvicidal tests with more than 20%
mortality in controls and pupae formed were
discarded and repeated again． If the control mortality
ranged between 5% and 20%，it was corrected using
Abbott’s formula (Abbott，1925)．
Corrected mortality =
(% Test mortality － % Control mortality)× 100
100 － % Control mortality
The data were subjected to regression analysis
using computerized SPSS 11． 5 Programme． The
7 期 Radhika WARIKOO et al．:Larvicidal potential of pine and cinnamon oils against Aedes aegypti 795
LC30，LC50，LC70，LC90 and LC99 values with 95%
fiducial limits were calculated in each bioassay to
measure difference between the test samples． The
results obtained with different oils were analyzed
using Student’s t-test with statistical significance
considered for P≤0． 05．
3 RESULTS
3． 1 Larvicidal bioassay
The larvicidal activities of the pine and
cinnamon oil，commercially available in New Delhi，
India，on early 4th instar larvae of Ae． aegypti are
presented in Table 1 and 2． Bioassays performed
with the two kinds of oils revealed their potency to
kill mosquito larvae at quite low concentrations． The
treatment resulted in complete mortality with no
pupal or adult emergence． The control or untreated
group did not exhibit any mortality within 24 h and
the larvae developed into pupae and then adults
within 48 － 72 h．
Our investigations clearly showed that at LC30 to
LC50 levels，the pine oil was 1． 8 to 2． 5-fold more
effective against the larvae of Ae． aegypti than the
cinnamon oil exhibiting an LC30 value of 0． 201 mg /L
and an LC50 value of 0． 330 mg /L (Table 1)． These
values，however，obtained with cinnamon oil were
0． 513 mg /L and 0． 613 mg /L，respectively (Table
2)． Likewise，at LC70 pine oil proved to be almost
1． 5 times more potent than cinnamon oil． However，
statistical analysis clearly showed that the LC values
obtained with pine oil treatment were significantly
different except at LC99 whereas the values obtained
after larvicidal bioassay with cinnamon oil were not
significantly different suggesting pine oil to be more
efficient than cinnamon oil． Nevertheless，the results
clearly show a gradually increasing effectiveness of
cinnamon oil than the pine oil with the increase in
lethal concentration (Fig． 1)． It is clear from the
Fig． 1 that the potency of cinnamon oil surpassed
that of pine oil at a concentration level somewhere
between LC85 and LC90 ． It was found to be 1． 06-fold
more effective than pine oil at LC90 while it became
1． 9-fold more potent at LC99 level．
Table 1 Larvicidal effects of pine oil on the early 4th instar larvae of Aedes aegypti
Concentration values LC30(mg /L) LC50(mg /L) LC70(mg /L) LC90(mg /L) LC99(mg /L) χ2(df) Regression equation S． E．
Lethal concentration 0． 20103 0． 33093 0． 54476 1． 11879 3． 02017
Lower fiducial limit 0． 12767 0． 25486 0． 43907 0． 80115 1． 70648 3． 676(3) Y = 1． 163 + 2． 422X 0． 210
Upper fiducial limit 0． 25997 0． 40915 0． 74616 2． 13553 9． 81049
Table 2 Larvicidal effects of cinnamon oil on the early 4th instar larvae of Aedes aegypti
Concentration values LC30(mg /L) LC50(mg /L) LC70(mg /L) LC90(mg /L) LC99(mg /L) χ2(df) Regression equation S． E．
Lethal concentration 0． 51315 0． 63159 0． 77736 1． 04915 1． 58680
Lower fiducial limit 0． 44934 0． 55608 0． 67215 0． 86589 1． 20983 0． 426(3) Y = 1． 160 + 5． 814X 0． 289
Upper fiducial limit 0． 58448 0． 74222 0． 96501 1． 43880 2． 53451
Fig． 1 Comparison of lethal concentrations of pine oil and
cinnamon oil against the early 4th instar larvae of Aedes aegypti
3． 2 Behavioural study
The symptomatological observations on the
larvae treated with the oils at LC99 revealed a
particular pattern in their behaviour (Table 3)． The
noticeable observation was the earlier appearance of
symptoms in cinnamon-treated larvae than the pine-
treated larvae． After immediate exposure to both the
oils， all larvae were active with clearly visible
movements． Initial abnormal excitation，
restlessness，and sluggishness were observed after
2 － 4 min of exposure which continued and persisted
till 10 － 35 min after which other abnormal motions
such as a coiling movement were seen． In both
cases，the treated larvae frequently sank down and
floated up again quickly． Thereafter more toxic
symptoms including tremor and convulsion appeared
followed by paralysis． More than one-half of the
larvae were paralyzed and had sunk to the bottom of
the bowl after 2 － 3 h． Moribund or dead larvae were
796 昆虫学报 Acta Entomologica Sinica 54 卷
Table 3 Symptomatological observations on the early 4th instar larvae of Aedes aegypti after
treatment with cinnamon oil and pine oil at LC99 level
S． no． Symptoms observed in the larvae treated at LC99 of the oil
Time of initiation of symptoms
Cinnamon oil
(LC99 = 3． 02017 mg /L)
Pine oil
(LC99 = 1． 58680 mg /L)
1 Initial abnormal excitation，restlessness，and sluggishness 2 min of exposure 4 min of exposure
2 Abnormal motions such as a coiling movement，quick wriggling action 10 － 20 min of exposure 30 － 35 min of exposure
3 Toxic symptoms with little tremor and convulsions 20 － 25 min of exposure 35 － 40 min of exposure
4 Excitation，restlessness，tremors and convulsions followed by paralysis 40 min of exposure 60 min of exposure
5 50% of the larvae paralyzed and sank to the bottom of the bowl 2 h of exposure 3 h of exposure
6 100% mortality 24 h of exposure 24 h of exposure
increasingly found each hour leading to 100% mortality
after 24 h．
3． 3 Morphological study
The morphological alterations of treated 4th
instar larvae，when observed revealed that all of the
organs，except anal gills，had a normal structural
appearance． Under light both treated and control
larvae showed similarities in morphological
architecture and cuticular sculpturing of the body
segments， and other organs such as the eyes，
antennae，mouthbrushes， setae， saddle， siphon，
and ventral brushes． However，the anal gills of all
treated larvae showed a structural alteration in the
internal structures with distinct shrinkage，while the
external features were normal in appearance (Fig．
2)．
Fig． 2 Light micrograph of anal gills of oil-treated
Aedes aegypti larvae showing normal external appearance
and shrinkage in internal structure
4 DISCUSSION
4． 1 Larvicidal bioassay
Essential oils extracted from different plants
have been reported to have larvicidal and repellent
properties against Ae． aegypti (Cavalcanti et al．，
2004;Amer and Mehlhorn 2006;Morais et al．，
2006;Silva et al．，2008;Waliwitiya et al．，2009)．
However，very few reports are available about the
potential of pine and cinnamon oil as mosquito
larvicide agent． The present investigations were
performed to assess the potential of commercially
available pine oil and cinnamon oil as larvicidal
agent against Ae． aegypti．
Our studies revealed the pine oil as the efficient
larvicidal agent against Ae． aegypti． On exposure to
commercially available pine oil，the early 4th instar
larvae showed an LC50 value of 0． 330 mg /L which
significantly increased to 1． 118 mg /L at LC90 ．
Similar results were reported by Ansari et al．
(2005)who also found the commercially available
pine oil highly effective against an Indian strain of
Ae． aegypti though the LC50(82． 1 mg /L)and LC90
value (252 mg /L)accounted by them were much
higher than obtained by us． They established the
larvicidal potential of pine oil against Cx．
quinquefasciatus (LC50:85． 7 mg /L;LC90:283． 4
mg /L) and An． stephensi (LC50:112． 6 mg /L;
LC90:329． 5 mg /L)too，though the efficacy was
found to be less than that against Ae． aegypti． They
further suggested that despite of larvicidal potential
of pine oil，it would not be practical to use it as a
larvicide in non-potable water in large breeding
habitats as it requires very high doses to be effective，
however，it might be used as larvicide selectively in
small breeding places such as in domestic and peri-
domestic containers， desert coolers， etc．， where
water is stagnant． On the other hand，our studies
have revealed quite low doses of pine oil to be
effective against the dengue fever mosquito larvae
and thus it can be used in large breeding habitats．
Still it is advisable that before reaching any
conclusion field experiments need to be conducted．
Similar results were found when the early fourth
instar larvae of Ae． aegypti were tested with
cinnamon oil． An interesting observation was that at
lower LC values，pine oil was found more effective
7 期 Radhika WARIKOO et al．:Larvicidal potential of pine and cinnamon oils against Aedes aegypti 797
(2． 5-fold at LC30，1． 8-fold at LC50)than cinnamon
oil，while gradually more efficacy was exhibited by
cinnamon oil (1． 06-fold at LC90)． Further research
is needed to find out the reasons for this finding．
Cheng et al． (2004)compared the leaf essential oils
from eight provenances of indigenous cinnamon
(Cinnamomum osmophloeum Kaneh．)and reported
that the essential oils of cinnamaldehyde type and
cinnamaldehyde /cinnamyl acetate type had an
excellent inhibitory effect against the 4th instar
larvae of Ae． aegypti with LC50 values of 36 mg /L
and 44 mg /L;and LC90 values of 79 mg /L and 85
mg /L，respectively． Cinnamaldehyde was reported
to possess the best mosquito larvicidal activity against
Ae． aegypti，with an LC50 of 29 mg /L and LC90 of 48
mg /L (Cheng et al．，2004)． The larvicidal activity
of cinnamon and other oils were recorded by Zhu et
al． (2006， 2008) against 4th instars of Ae．
albopictus，Ae． aegypti，and Culex pipiens pallens．
4． 2 Behavioural study
The gradual abnormal behaviour in the motion
of treated larvae followed by excitation，convulsions，
paralysis，and then resulting in 100% kill indicates
a delayed type of larval killing from the plant oils． In
fact，pine oil proved to have a more delayed effect in
comparison to the cinnamon oil． These observations
correspond to the earlier studies carried out to
evaluate the larvicidal potential of plant-derived
materials against some mosquito species (Choochote
et al．，2004，2005;Dharmagadda et al．，2005;
Chaithong et al．，2006)． These studies conducted
with plant oils or extracts of Kaempferia galanga，
Tagetes patula， Apium graveolens， Curcuma
aromatica， and Piper species showed similar
symptoms in the exposed larvae though the time for
the appearance of the toxic symptoms was relatively
different． The observed symptoms being similar to
those of nerve poisons，though slow，suggest the
probable toxic effect of the plants on the
neuromuscular system．
4． 3 Morphological study
Our studies also showed the shrinkage in the
internal membrane of the anal gills of the Ae． aegypti
larvae treated with both the oils． These investigations
are similar to that of Chaithong et al． (2006)who
reported the remarkable shrinkage in the internal
structure of anal papillae in the larvae of a Thailand
strain of Ae． aegypti when treated with ethanolic
extracts of black pepper，while most of the other
organs of dead larvae had a normal appearance．
Likewise，Cx． quinquefasciatus larvae treated with
ethanolic extract of Kaempferia galanga revealed the
severely morphological disruption of anal papillae
with a shrunken cuticle border and destroyed surface
with loss of ridge-like reticulum under light and
scanning electron microscopy，respectively (Insun et
al．，1999)． Green et al． (1991) also reported
swollen anal papillae in Ae． aegypti larvae after
treatment with whole oil of Tagetes minuta． It was
suggested that structural deformation of anal papillae
probably led to their dysfunction，which may be
intrinsically associated with the death of mosquito
larvae (Chaithong et al．，2006)． Earlier，it was
reported that uptake and elimination of most ions in
mosquito larvae occur via the anal papillae，which
was markedly reduced or lost in papilla-less larvae
(Garrett and Bradley，1984;Clements，1992)．
This indicated that the lack or dysfunction of the anal
papillae probably led to an interruption of the
osmotic and ionic regulation． However， the
microscopic study alone cannot ascertain the cause of
the larvicidal effects of the oils，which may be at
physiological or biochemical level． Extensive and
further research is required to investigate the site of
action of the oils．
4． 4 Conclusion
Our studies prove the potential of pine oil and
cinnamon oil as the larvicidal agent against Ae．
aegypti． The pine oil proved to be apparently more
effective at lower LC values while gradually
cinnamon oil proved to be more potent than pine oil．
The toxic symptoms and deformity in anal gills
suggest different modes of action of oils． Further
research is needed to explore the action site of oils．
The studies suggest that a formulation using the
bioactive constituent of investigated oils could be
used against mosquito larvae without the potential for
adverse health effects and an added bonus of a
pleasant smell． Further investigations are required to
identify the bioactive constituents present in these
oils possessing mosquicidal properties．
ACKNOWLEDGEMENTS The authors are highly grateful to
University Grants Commission for providing financial assistance
to carry out the present investigations． Thanks are extended to
Dr． Savithri Singh，Principal，Acharya Narendra Dev College for
providing infrastructure and research facilities．
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7 期 Radhika WARIKOO et al．:Larvicidal potential of pine and cinnamon oils against Aedes aegypti 799
市售长叶松油和锡兰肉桂油对登革热媒蚊
埃及伊蚊的灭幼效果
Radhika WARIKOO，Naim WAHAB，Sarita KUMAR*
(Department of Zoology，Acharya Narendra Dev College，University of Delhi，Kalka Ji，New Delhi 110019，India)
摘要:松油和桂皮油由于具有芳香性气味，因而成为良好的成虫驱避剂，但是关于它们对蚊虫的杀幼虫作用研究
不多。为揭示市售的长叶松 Pinus longifolia 油和锡兰肉桂 Cinnamomum zeylanicum 油对来源于印度德里的埃及伊
蚊 Aedes aegypti 4 龄幼虫的毒杀潜力，我们进行了室内研究，以幼虫死亡率及行为改变和形态改变等指标评估其
杀幼虫潜力。结果表明:两种油对埃及伊蚊 4 龄幼虫均具有毒杀作用，且松油的灭幼效果优于桂皮油。松油的
LC50和 LC70值分别为 0． 33093 mg /L 和 0． 54476 mg /L，而桂皮油的 LC50和 LC70值分别为 0． 63159 mg /L和 0． 77736。
进一步观察发现，LC90剂量下桂皮油的杀幼虫潜力强于松油，其 LC90为 1． 11879 mg /L，而松油的 LC90为 1． 04915
mg /L。在处理的幼虫中观察到行为改变，如兴奋、坐立不安、颤抖、痉挛然后瘫痪，说明这两种油可能对其神经
肌肉系统产生了影响。显微观察处理幼虫的形态改变发现，与对照相比，大多数器官的外观正常，只是肛鳃略为
内收缩而引起结构畸形，提示肛腮可能是这两种油的作用位点，腮的功能异常引起了幼虫死亡。这两种油品可开
发用作防治蚊虫的新型杀幼虫药剂。
关键词:埃及伊蚊;松树;肉桂;杀幼虫潜力;肛腮;痉挛
中图分类号:Q965． 9 文献标识码:A 文章编号:0454-6296(2011)07-0793-07
(责任编辑:赵利辉)