Larvicidal effect of pepper plants on Aedes aegypti (L.

) (Diptera: Culicidae)
Author(s): Udom Chaithong, Wej Choochote, Kittichai Kamsuk, Atchariya Jitpakdi, Pongsri
Tippawangkosol, Dana Chaiyasit, Daruna Champakaew, Benjawan Tuetun, and Benjawan Pitasawat
Source: Journal of Vector Ecology, 31(1):138-144.
Published By: Society for Vector Ecology
DOI: http://dx.doi.org/10.3376/1081-1710(2006)31[138:LEOPPO]2.0.CO;2
URL: http://www.bioone.org/doi/full/10.3376/1081-1710%282006%2931%5B138%3ALEOPPO
%5D2.0.CO%3B2

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138 Journal of Vector Ecology June 2006

Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae)

Udom Chaithong , Wej Choochote, Kittichai Kamsuk, Atchariya Jitpakdi, Pongsri Tippawangkosol,
Dana Chaiyasit, Daruna Champakaew, Benjawan Tuetun, and Benjawan Pitasawat

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

Received 9 November 2005; Accepted 25 February 2006

ABSTRACT: Ethanolic extracts derived from three species of the Piperaceae (pepper) family, Piper longum L., P. ribesoides
Wall., and P. sarmentosum Roxb. ex Hunt., were evaluated for efficacy against early 4th instar larvae of Aedes aegypti
mosquitoes using larvicidal bioassays. The highest larvicidal efficacy was established from P. longum, followed by P.
sarmentosum and P. ribesoides, with LC50 values of 2.23, 4.06, and 8.13 ppm, respectively. Observations of morphological
alterations on treated 4th instar larvae revealed that most organs, except anal papillae, had a normal structural appearance that
was similar to controls. Under light microscopy, the internal structures of anal papillae in the treated larvae showed shrinkage,
while the external features were normal in appearance. Ultrastructural studies, however, clearly demonstrated external
destruction, with extensive damage and shrunken cuticle of the anal papillae. The structural deformation of anal papillae
probably led to their dysfunction, which may be intrinsically associated with the death of the larvae. This study affords some
evidence regarding the action site of the pepper extracts and suggests their potential in developing new types of larvicides
used for mosquito control. Journal of Vector Ecology 31 (1): 138-144. 2006.

Keyword Index: Piperaceae, pepper, Piper longum, Piper ribesoides, Piper sarmentosum, Aedes aegypti, larvicide.

INTRODUCTION
The medical importance of mosquitoes as vectors for the
transmission of serious diseases that cause morbidity,
mortality, economical loss, and social disruption such as
malaria, lymphatic filariasis, and viral diseases is well
documented (Becker et al. 2003). Aedes aegypti, the primary
carrier for viruses that cause dengue and dengue hemorrhagic
fever and yellow fever, are widespread over large areas of the
tropics and subtropics. At present, no effective vaccine is
available for dengue, therefore, the only way of reducing the
incidence of this disease is by mosquito control, which is
frequently dependent on applications of conventional synthetic
insecticides (Malavige et al. 2004). Chemical measures in
public health programs were initially considered likely to
decrease mosquito populations, but these have failed because
the constant use of chemical insecticides has often led to the
disruption of natural biological control systems and outbreaks
of insect species. Moreover, problems created by using
synthetic insecticides include the development of mosquito
resistance, environmental pollution, and undesirable effects
on humans, mammals, and other non-target organisms (Brown
1986, Lee et al. 2001). In an attempt to resolve these problems,
attention to insecticides of natural origin, particularly plant-
derived products, has been recently revived. A considerable
number of studies have emphasized the research and
development of herbal substances for controlling mosquitoes
(Sukumar et al. 1991, Tsao et al. 2002, Jeyabalan et al. 2003).
Although results vary, natural plant products may be a possible
alternative to synthetic substances, as they are effective and
compatible with human and animal life and the environment.
The Piperaceae (pepper) family contains approximately
2,000 species, which are widely grown and commonly used
in tropical regions as medicines, spice, and condiments in
regional cuisine (Numba 1993, Shultes and Raffauf 1990).
Pepper plants have also been prescribed for pest control as
they contain potentially insecticidal compounds (Freeborn and
Wymore 1929, Lathrop and Keirstead 1946, Su and Horvat
1981). Some Piper spp., Piper longum, P. nigrum, P.
guanacastensis, and their bioactive constituents are reported
to have remarkably larvicidal activity against various mosquito
species such as Culex pipiens pallens, Ae. aegypti, Ae. togoi,
and Ae. atropalpus (Pereda-Miranda et al. 1997, Lee 2000,
Park et al. 2002). The aim of this study was to evaluate the
potential of three pepper plants against the larvae of Ae.
aegypti through larvicidal bioassays and observe the
morphological alterations of larvae treated with a lethal dosage
of the pepper extracts under light and scanning electron
microscopy.
MATERIALS AND METHODS
Plant extraction
Three Piper species of the Piperaceae family, Piper
longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex
Hunt., were obtained from E.A.R. Samunpri, a commercial
supplier in Chiang Mai province, northern Thailand. The
voucher specimens were preserved at the Department of
Parasitology, Faculty of Medicine, Chiang Mai University.
Dried and powdered material of each plant (1 kg) was
successively extracted three times by maceration with 3 liters
of 95% ethanol at room temperature for 7 days. The crude
extracts were separated by suction filtered through a Bchner
funnel, and the combined filtrates were concentrated to dryness
Vol. 31, no. 1 Journal of Vector Ecology
with a rotary evaporator at 60 C until the solvent completely
evaporated. The ethanolic extract of each plant was thus
obtained, lyophilized, and then refrigerated at -20 C until
testing for mosquitocidal activity.
Mosquitoes
A laboratory colony of Ae. aegypti mosquitoes, originally
collected in Chiang Mai province, was colonized continuously
for over 20 generations in a laboratory free of exposure to
pathogens and insecticides at the Department of Parasitology,
Faculty of Medicine, Chiang Mai University. The adults were
reared in a room maintained at 25-30 C and 80-90% relative
humidity under a photoperiod of 14:10 h (light/dark). In
addition to 10% sucrose and 10% multivitamin syrup (SEVEN
SEAS ), adult females were periodically blood-fed on
restrained rats to obtain protein for egg maturation. Finely
ground dog biscuit was used as a food source for the larvae.
Under these conditions, the life cycle of this mosquito from
egg to adult took about 3-4 weeks. Early 4th instar larvae were
used in the experiments.
Larvicidal bioassay
The test for the larvicidal effect of ethanolic extract
derived from Piper spp. against Ae. aegypti was conducted
in accordance with the WHO standard method (WHO 1981).
Each pepper extract was dissolved in absolute ethanol or
dimethylsulphoxide (DMSO) to prepare a graded series of
concentrations. Batches of 25 early 4th instar larvae of Ae.
aegypti were transferred in 25 ml of distilled water to a 500
ml enamel bowl containing 224 ml of distilled water and 1.0
ml of a serial dilution of each plant extract. Four replicate
tests were carried out simultaneously, with a final total of 100
larvae for each concentration. The toxicity of each plant extract
was evaluated with four to six concentrations yielding a range
of 0-100% mortality. Controls received ethanol- or DMSO-
distilled water, while the untreated larvae were maintained in
distilled water only. After treatment, symptoms in treated
larvae were observed and recorded immediately and at time-
intervals, and no food was offered to the larvae. The larvae
were considered dead if, at the end of 24 h, they showed no
sign of swimming movements even after gentle touching with
a glass rod, as described in the World Health Organizations
technical report series. The dead larvae in four replicates were
combined and expressed as a percentage mortality of each
concentration. Bioassays were performed at 25-30 C,
replicated three times with mosquitoes from different rearing
batches, and reported for the average of individual test results.
Light and scanning electron microscopy
After treatment with a lethal dosage (LC99) of each pepper
extract, the dead larvae were studied for morphological
alterations under light and scanning electron microscopy.
Larvae mounted with Hoyers medium on a microscope slide
were scrutinized under light microscopy. Morphological
changes in body segments including the head, thorax, and
abdomen, and other organs such as the eyes, antennae,
mouthbrushes, setae, saddle, and anal gills were observed,
photographed, and compared with those of the controls. For
139
scanning electron microscopic study using low vacuum, the
treated larvae were submerged in liquid nitrogen for a few
minutes and then attached to aluminum stubs with double-
stick tape. Ultrastructural details were observed and
photographs taken with SEM (JEOL JSM-5910LV).
Statistical analysis of data
It was necessary to obtain not less than three mortality
counts of between 10% and 90%. Experimental tests with
more than 20% control mortality were discarded and then
repeated. However, when the control mortality ranged from
5-20%, the percentage mortality observed (%M) was
corrected by Abbotts formula (Abbott 1925):
%M = % test mortality -% control mortality x 100
100-% control mortality
Dosage-mortality lines were estimated by a computerized
log-probit analysis (Harvard Programming; Hg1, 2). The 95%
confidence intervals (CI) at the lethal dosage of 50% and 95%
(LD50 and LD95) were used to measure differences between
test samples.
RESULTS AND DISCUSSION
Ethanolic extractions of the three Piper species, P.
longum, P. ribesoides, and P. sarmentosum, provided semi-
solid materials with various strengths of odor and average
yields of 8.89, 3.21, and 5.30% (w/w), respectively (Table
1). In this study, early 4th instar larvae of Ae. aegypti, under
laboratory conditions, were subjected to a dose-dependent
efficacy of the three Piper extracts (Table 2). Following
treatment of increasing dosages of P. longum, P. sarmentosum,
and P. ribesoides from 1.5-3.5, 2-10, and 6-12 ppm,
respectively, the larval mortality increased from 20.3-96.3,
12.3-97.7, and 16.3-90.0%, respectively. No pupal or adult
emergence was observed in the treatment, as complete
mortality occurred. In the case of the control or untreated
group, mortality observed within 24 h was zero and the larvae
developed into pupae and then adults within 48-72 h. Among
the extracts, P. longum showed more remarkable larvicidal
potential than P. sarmentosum and P. ribesoides, presenting
LC50 and LC95 values of 2.23 and 4.80, 4.06 and 12.06, and
8.13 and 14.01 ppm, respectively. Variety of types and levels
of active constituents in each Piper species may be responsible
for the variability in their potential against Ae. aegypti.
Symptomatological observations on the larvae treated
with the three Piper extracts revealed a similar manner of
toxicity in the larvae. All larvae were still active immediately
after exposure to LC99 of each pepper extract, and the feeding
process and normal zigzag motion of these treated larvae were
clearly seen. However, after 5 min of exposure, abnormal
evidence of excitation, restlessness, and sluggishness was
initially observed. Excitation and restlessness persisted for
between 10-30 min, and other anomalous motions were seen
such as a coiling movement. The treated larvae frequently
sank down and floated up again quickly. During the period of
30-60 min, some larvae showed more toxic symptoms
140 Journal of Vector Ecology June 2006

Table 1. Physical characteristics and percentage yields of ethanolic extracts of the Piper species.

Botanical name Physical characteristics Yield

English name Part used
(Voucher) Appearance Color Odor (%)
P. longum
Long pepper Fruit Semi-solid Dark brown Sweet-spicy 8.89
(PARA-PI-001/1)
P. ribesoides
Jakhang pepper Wood Semi-solid Dark brown Raw smell 3.21
(PARA-PI-002/1)
P. sarmentosum
Variegatum Whole plant Semi-solid Dark green Sweet-herbal 5.30
(PARA-PI-003/1)

Figures 1-4. Light (1, 2) and scanning electron (3, 4) micrographs of anal gills of Ae. aegypti larvae. (1) Control larva
showing two pairs of normal gills with sac-like structure covered by smooth cuticle. (2) Pepper-treated larva showing four
anal gills with externally normal appearance but internally shrunken structure (arrow). (3) Control larva showing cone-
shaped normal gills with intact cuticle. (4) Pepper-treated larva showing damage and shrunken cuticle of anal papillae.
Vol. 31, no. 1 Journal of Vector Ecology 141

Table 2. Larvicidal activity of the ethanolic extracts derived from three Piper species against 4th instar larvae of Ae. aegypti.
Piper species extract % Mortality Larvicidal activity
(ppm) (MeanSE) (95% C.I., ppm)
LD50 LD95 LD99
P. longum 2.23 4.80 7.38
1.5 20.254.60 (2.11-2.37) (4.10-6.18) (5.82-10.85)
2.0 34.509.71
2.5 62.7510.18
3.0 76.253.90
3.5 96.252.38
Control 0
Untreated 0
P. ribesoides 8.13 14.01 19.00
6 16.2511.73 (7.84-8.42) (12.87-15.78) (16.71-22.80)
7 31.7519.02
8 45.0022.38
9 67.2521.82
10 72.5017.54
12 90.009.09
Control 0
Untreated 0
P. sarmentosum 4.06 12.06 22.20
2 12.3310.21 (3.68-4.43) (10.05-15.69) (16.84-33.40)
4 51.008.54
6 72.0011.53
8 87.005.57
10 97.670.58
Control 0
Untreated 0

including tremor and convulsion at the bottom of the enamel
bowl. Afterwards, more and more larvae exhibited the toxic
symptoms. Appearance of excitation, restlessness, tremors,
and convulsions followed by paralysis was clearly seen. Two
h after treatment, more than one-half of the larvae were
paralyzed and had sunk to the bottom of the bowl. Moribund
or dead larvae were increasingly found from 2 to 7 h. At the
end of a 24-h exposure period, all larvae had subsequently
died. The apparent results indicated a delayed type of larval
killing from these Piper species. Our findings corresponded
to those of earlier studies (Insun et al. 1999, Dharmagadda et
al. 2005, Choochote et al. 2004, 2005), which were carried
out to evaluate the larvicidal potential of plant-derived
materials against some mosquito species. Although symptoms
of larvae exposed to the plant oils or extracts, Kaempferia
galanga, Tagetes patula, Apium graveolens, Curcuma
aromatica, and Piper species seem to be similar, the times to
their showing toxic symptoms were relatively different.
Similarity in the behavior of larvae treated with these plants
to those of nerve poisons, i.e. excitation, convulsions,
paralysis, and death, indicated that the plants probably had a
toxic effect on the neuromuscular system (Sakthivadivel and
Thilagavathy 2003, Choochote et al. 2004). Nevertheless, the
symptoms caused by nerve poisons happened more rapidly
than those observed in the plant-treated larvae.
Observations on the morphological alterations of treated
4th instar larvae revealed that most organs, except anal papillae
(gills), had a normal structural appearance. Under light and
scanning electron microscopes, both treated and control larvae
showed similarities in morphological architecture and
cuticular sculpturing of the head, thorax, and abdomen
segments, and other organs such as the eyes, antennae,
mouthbrushes, setae, saddle, siphon, and ventral brushes. A
distinct difference, however, was the structural alteration of
the anal gills observed in the pepper-treated larvae. This organ
is involved in the regulation of electrolyte levels. Under light
microscopy (Figures 1 and 2), the internal structures of the
anal papillae in treated larvae showed remarkable shrinkage,
while the external features were normal in appearance.
Ultrastructural studies (Figures 3 and 4), however, clearly
demonstrated external destruction, with extensive damage and
a shrunken cuticle of the anal papillae. These results therefore
indicated that the toxic effect of pepper extract is
predominantly on the anal papillae, leading to their
morphological deformation. These findings corresponded to
those of earlier works that investigated the effect of plant-
derived compounds on some species of mosquitoes. Studies
conducted by Insun et al. (1999) revealed the severely
142 Journal of Vector Ecology
morphological disruption of anal papillae observed in dead
Cx. quinquefasciatus larvae. After treatment with ethanolic
extract of K. galanga, damaged anal papillae, with a shrunken
cuticle border and destroyed surface with loss of ridge-like
reticulum were found under light and scanning electron
microscopes, respectively. Green et al. (1991) reported distinct
features of alteration such as highly swollen anal papillae of
Ae. aegypti larvae after treatment with whole oil of Tagetes
minuta. Structural deformation of anal papillae probably led
to their dysfunction, which may be intrinsically associated
with the death of mosquito larvae.
The two pairs of anal papillae are flexible, sac-like
structures consisting of an epithelium covered by cuticle and
situated on an extension of the terminal segment of mosquito
larvae. In the fresh-water mosquito larvae, uptake and
elimination of most ions occur via the anal papillae, while the
process of ion conservation is mainly located in the alimentary
canal (Garrett and Bradley 1984, Clements 1992). The
capacity to take up sodium, potassium, chloride, and
phosphate ions from the medium was markedly reduced or
lost in papilla-less larvae (Koch 1938, Hassett and Jenkins
1951, Ramsay 1953). This indicated that the lack or
dysfunction of the anal papillae probably led to an interruption
of the osmotic and ionic regulation. Although the lower limits
of ion concentrations that permit survival of mosquito larvae
have not yet been established, ionic imbalance resulting from
the interruption of ionic regulation is also a harmful condition.
Considerable research on the larvicidal potential of
natural products for controlling Aedes mosquitoes has been
carried out, but with varied results. Essential oils extracted
from nine plants commonly found in northeastern Brazil
exhibited various degrees of larvicidal activity against Ae.
aegypti, with LC50 values that ranged from 60 to 538 ppm
(Cavalcanti et al. 2004). Ethanolic extracts from fruit
endocarps of Melia azedarach and Azadirachta indica, two
members of the family Meliaceae, were found to have lethal
effects on Ae. aegypti larvae, with LC50 values ranging from
0.017 to 0.034 g% (Wandscheer et al. 2004). The effective
constituents in leaf essential oils of Cinnamomum
osmophloeum Provenances including cinnamaldehyde,
eugenol, anethole, and cinnamyl acetate showed an inhibitory
effect against the 4th instar larvae of Ae. aegypti, with LC50
values of lower than 50 ppm (Cheng et al. 2004). The LC50 of
cinnamaldehyde, which had the strongest larvicidal activity,
was 29 ppm. Larvicidal determination of seventeen Brazilian
plants demonstrated that five oils derived from Anacardium
occidentalis, Copaifera langsdorffii, Carapa guianensis,
Cymbopogon winterianus, and Ageratum conyzoides, and one
ethanolic extract of Annona glabra showed high activities
against Ae. aegypti larvae, with LC50 values of 14.5, 41, 57,
98, 148, and 27 mg/l, respectively (de Mendona et al. 2005).
Comparison with the results mentioned above reveals that
the larvicidal potential of Piper spp. tested in this study was
greater than or comparable to that of previously described
natural products.
Recently, there has been a growing interest in plants
belonging to the family Piperaceae as a potential source of
bioactive chemical compounds against mosquito vectors. Park
June 2006
et al. (2002) reported an effect of larvicidal constituents
isolated from P. nigrum fruit against the three mosquito
species, Cx. pipiens pallens, Ae. aegypti, and Ae. togoi.
Larvicidal activity against Ae. aegypti was more marked in
retrofractamide A (0.039 ppm) than quineensine (0.89 ppm),
pellitorine (0.92 ppm), and pipercide (0.1 ppm). In the study
of Yang et al. (2002), one active isolated from the hexane
fraction of the P. longum fruit, pipernonaline, showed potent
larvicidal activity against Ae. aegypti with LC50 of 0.25 mg/l.
These promising Piper-derived larvicides are results of the
search for new phytochemical agents from Piperaceae plants,
which should influence further research of other plants
belonging to this family in order to find affordable natural
substances for use in mosquito control. Although the larvicidal
potential of crude ethanolic extracts derived from the three
Piper species observed in this study was significantly lower
than that in previous works, further studies for the isolation
and identification of bioactive compounds would be useful
in developing new types of mosquito larvicides.
Investigations on the toxic effect and mode of action of
bioactive compounds against mosquito larvae were previously
carried out. Utrastructural observations on Ae. aegypti larvae
after treating with ethanolic extract of Derris urucu roots
revealed an imperfect peritrophic matrix and extensive
damage of the midgut epithelium (Gusmo et al. 2002).
Correspondingly, the toxic effect of ethanolic-extracted
Magonia pubescens on Ae. aegypti larvae was mainly in the
midgut, showing partial or total cell destruction, high
citoplasmatic vacuolization, increased subperitrophic space
and cell hypertrophy, and the epithelium did not maintain its
monolayer appearance (Arruda et al. 2003). Generally,
insecticides enter insects by cuticular penetration and/or
ingestion and then pass throughout the interior of the insect
to the site of action (Matsumura 1975). Although these reports
provided some evidence regarding the action site of these
bioactive compounds, the mechanism causing mortality of
mosquito larvae is still unknown and needs to be studied
further. However, this study demonstrated and emphasized
the potential of Piperaceae plants against Ae. aegypti larvae
and its benefit to developing new types of larvicides used for
mosquito control.
Acknowledgments
The authors acknowledge the staff members of the
Department of Parasitology, Faculty of Medicine, Chiang Mai
University for their cooperation. We thank Budsabong
Kuntalue and the staff of the Electron Microscopy Research
and Service Center (EMRSC), Chiang Mai University for their
technical assistance. Acknowledgment is extended to the
Faculty of Medicine for its financial support of this research
project and to the Faculty of Medicine Endowment Fund for
Research Publication for its financial support in publishing
this paper.
Vol. 31, no. 1 Journal of Vector Ecology
REFERENCES CITED
Abbott, W.S. 1925. A method of computing the effectiveness
of an insecticide. J. Econ. Entomol. 18: 265-266.
Arruda, W., G.M.C. Oliveira, and E.G. da Silva. 2003. Toxicity
of the ethanol extract of Magonia pubescens on larvae
Aedes aegypti. Rev. Soc. Bras. Med. Trop. 36: 17-25.
Becker, N., D. Petri, M. Zgomba, C. Boase, C. Dahl, J. Lane,
and A. Kaiser. 2003. Mosquitoes and their control. New
York: Kluwer Academic/Plenum Publishers.
Brown, A.W.A. 1986. Insecticide resistance in mosquitoes:
pragmatic review. J. Am. Mosq. Contr. Assoc. 2: 123-
140.
Cavalcanti, E.S.B., S.M. de Morais, M.A.A. Lima, and E.W.P.
Santana. 2004. Larvicidal activity of essential oils from
Brazilian plants against Aedes aegypti L. Mem. Inst.
Oswaldo Cruz 99: 541-544.
Cheng, S.S., J.Y. Liu, K.H.Tsai, W.J. Chen, and S.T. Chang.
2004. Chemical composition and mosquito larvicidal
activity of essential oils from leaves of different
Cinnamomum somophloeum Provenances. J. Agric. Food
Chem. 52: 4395-4400.
Choochote, W., B. Tuetun, D. Kanjanapothi, E.
Rattanachanpichai, U. Chaithong, P. Chaiwong, A.
Jitpakdi, P. Tippawangkosol, D. Riyong, and B. Pitasawat.
2004. Potential of crude seed extract of celery, Apium
graveolens L., against the mosquito Aedes aegypti (L.)
(Diptera: Culicidae). J. Vector Ecol. 29: 340-346.
Choochote, W., D. Chaiyasit, D. Kanjanapothi, E.
Rattanachanpichai, A. Jitpakdi, B. Tuetun, and B.
Pitasawat. 2005. Chemical composition and anti-
mosquito potential of rhizome extract and volatile oil
derived from Curcuma aromatica against Aedes aegypti
(Diptera: Culicidae). J. Vector Ecol. 30: 302-309.
Clements, A.N. 1992. The biology of mosquitoes. Vol. 1:
Development, nutrition and reproduction. Chapman and
Hall, London.
de Mendona, F.A.C., K.F.S. da Silva, K.K. dos Santos, K.A.L.
Ribeiro Jnior, and A.E.G. SantAna. 2005. Activities of
some Brazilian plants against larvae of the mosquito
Aedes aegypti. Fitoterapia 76: 629-636.
Dharmagadda, V.S.S., S.N. Naik, P.K. Mittal, and P.
Vasudevan. 2005. Larvicidal activity of Tagetes patula
essential oil against three mosquito species. Biores.
Technol. 96: 1235-1240.
Freeborn, S.B. and F.H. Wymore. 1929. Attempts to protect
sweet corn from infestations of the corn earworm,
Heliothis obsoleta (Fabr.). J. Econ. Entomol. 22: 666-
671.
Garrett, M. and T. J. Bradley. 1984. The pattern of osmotic
regulation in larvae of the mosquito Culiseta inornata. J.
Exp. Biol. 113: 133-141.
Green M.M., J.M. Singer, D.J. Sutherland, and C.R. Hibben.
1991. Larvicidal activity of Tagetes minuta (Marigold)
toward Aedes aegypti. J. Am. Mosq. Contr. Assoc. 7: 282-
286.
Gusmo, D.S., V. Pscoa, L. Mathias, I.J.C. Vieira, R. Braz-
Filho, and F.J.A. Lemos. 2002. Derris (Lonchocarpus)
143
urucu (Leguminosae) extract modifies the peritrophic
matrix structure of Aedes aegypti (Diptera: Culicidae).
Mem. Inst. Oswaldo. Cruz. 97: 371-375.
Hassett, C.C. and D. W. Jenkins. 1951. The uptake and effect
of radiophosphorus in mosquitoes. Physiol. Zool. 24:
257-266.
Insun, D., W. Choochote, A. Jitpakdi, U. Chaithong, P.
Tippawangkosol, and B. Pitasawat. 1999. Possible site
of action of Kaempferia galanga in killing Culex
quinquefasciatus larvae. Southeast Asian J. Trop. Med.
Publ. Hlth. 30: 195-199.
Jeyabalan, D., N. Arul, and P. Thangamathi. 2003. Studies on
effects of Pelargonium citrosa leaf extracts on malarial
vector, Anopheles stephensi Liston. Biores. Technol. 89:
185-189.
Koch, H.J. 1938. The absorption of chloride ions by the anal
papillae of Diptera larvae. J. Exp. Biol. 15: 152-160.
Lathrop, F.H. and L.G. Keirstead. 1946. Black pepper to
control the bean weevil. J. Econ. Entomol. 39: 534.
Lee, S.E. 2000. Mosquito larvicidal activity of pipernonaline,
a piperidine alkaloid derived from long pepper, Piper
longum. J. Am. Mosq. Contr. Assoc. 16: 245-247.
Lee, S.E., J.E. Kim, and H.S. Lee. 2001. Insecticide resistance
in increasing interest. Agric. Chem. Biotechnol. 44: 105-
112.
Malavige, G.N., S. Fernando, D.J. Fernando, and S.L.
Seneviratne. 2004. Dengue viral infections. Postgrad.
Med. J. 80: 588-601.
Matsumura, F. 1975. Toxicology of insecticides. Plenum Press,
New York.
Numba, T. 1993. The encyclopedia of Wakan-Yaku
(Traditional Sino-Japanese Medicine) with color
pictures, Vol II. Osaka: Hoikusha.
Park, I.K., S.G. Lee, S.C. Shin, J.D. Park, and Y.J. Ahn. 2002.
Larvicidal activity of isobutylamides identified in Piper
nigrum fruits against three mosquito species. J. Agric.
Food Chem. 50: 1866-1870.
Pereda-Miranda, R., C.B. Bernard, T. Durst, J.T. Arnason., P.
Snchez-Vindas, L. Poveda, and L. San Romn. 1997.
Methyl 4-hydroxy-3-(3-methyl-2-butenyl) benzoate,
major insecticidal principle from Piper guanacastensis.
J. Nat. Prod. 60: 282-284.
Ramsay, J.A. 1953. Exchanges of sodium and potassium in
mosquito larvae. J. Exp. Biol. 30: 79-89.
Sakthivadivel, M. and D. Thilagavathy. 2003. Larvicidal and
chemosterilant activity of the acetone fraction of
petroleum ether extract from Argemone mexicana L. seed.
Biores. Technol. 89: 213-216.
Shultes, R.E. and R.F. Raffauf. 1990. The healing forest:
medicinal and toxic plants of the Northwest Amazonia.
Portland: Dioscoride Press.
Su, H.C.F. and R. Horvat. 1981. Isolation, identification and
insecticidal properties of Piper nigrum amides. J. Agric.
Food Chem. 29: 115-118.
Sukumar, K., M.J. Perich, and L.R. Booba. 1991. Botanical
derivatives in mosquito control: a review. J. Am. Mosq.
Contr. Assoc. 7: 210-237.
Tsao, R., F.E. Romanchuk, C.J. Peterson, and J.R. Coats. 2002.
144 Journal of Vector Ecology
Plant growth regulatory effect and insecticidal activity
of extracts of tree of Heaven (Ailanthus altissima L.).
BMC. Ecol. 2: 1-8.
Wandscheer, C.B., J.E. Duque, M.A.N. da Silva, Y. Fukuyama,
J.L. Wohlke, J. Adelmann, and J.D. Fontana. 2004.
Larvicidal action of ethanolic extracts from fruit
endocarps of Melia azedarach and Azadirachta indica
against the dengue mosquito Aedes aegypti. Toxicon 44:
829-835.
June 2006
World Health Organization. 1981. Instructions for
determining the susceptibility or resistance of mosquito
larvae to insecticides. WHO/VBC/81.807.
Yang, Y.C., S.G. Lee, H.K. Lee, M.K. Kim, S.H. Lee, and
H.S. Lee. 2002. A piperidine amide extracted from Piper
longum L. fruit shows activity against Ae. aegypti
mosquito larvae. J. Agric. Food Chem. 50: 3765-3767.