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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 2,000 species, which are widely grown and commonly used
in tropical regions as medicines, spice, and condiments in
The medical importance of mosquitoes as vectors for the regional cuisine (Numba 1993, Shultes and Raffauf 1990).
transmission of serious diseases that cause morbidity, Pepper plants have also been prescribed for pest control as
mortality, economical loss, and social disruption such as they contain potentially insecticidal compounds (Freeborn and
malaria, lymphatic filariasis, and viral diseases is well Wymore 1929, Lathrop and Keirstead 1946, Su and Horvat
documented (Becker et al. 2003). Aedes aegypti, the primary 1981). Some Piper spp., Piper longum, P. nigrum, P.
carrier for viruses that cause dengue and dengue hemorrhagic guanacastensis, and their bioactive constituents are reported
fever and yellow fever, are widespread over large areas of the to have remarkably larvicidal activity against various mosquito
tropics and subtropics. At present, no effective vaccine is species such as Culex pipiens pallens, Ae. aegypti, Ae. togoi,
available for dengue, therefore, the only way of reducing the and Ae. atropalpus (Pereda-Miranda et al. 1997, Lee 2000,
incidence of this disease is by mosquito control, which is Park et al. 2002). The aim of this study was to evaluate the
frequently dependent on applications of conventional synthetic potential of three pepper plants against the larvae of Ae.
insecticides (Malavige et al. 2004). Chemical measures in aegypti through larvicidal bioassays and observe the
public health programs were initially considered likely to morphological alterations of larvae treated with a lethal dosage
decrease mosquito populations, but these have failed because of the pepper extracts under light and scanning electron
the constant use of chemical insecticides has often led to the microscopy.
disruption of natural biological control systems and outbreaks
of insect species. Moreover, problems created by using MATERIALS AND METHODS
synthetic insecticides include the development of mosquito
resistance, environmental pollution, and undesirable effects Plant extraction
on humans, mammals, and other non-target organisms (Brown Three Piper species of the Piperaceae family, Piper
1986, Lee et al. 2001). In an attempt to resolve these problems, longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex
attention to insecticides of natural origin, particularly plant- Hunt., were obtained from E.A.R. Samunpri, a commercial
derived products, has been recently revived. A considerable supplier in Chiang Mai province, northern Thailand. The
number of studies have emphasized the research and voucher specimens were preserved at the Department of
development of herbal substances for controlling mosquitoes Parasitology, Faculty of Medicine, Chiang Mai University.
(Sukumar et al. 1991, Tsao et al. 2002, Jeyabalan et al. 2003). Dried and powdered material of each plant (1 kg) was
Although results vary, natural plant products may be a possible successively extracted three times by maceration with 3 liters
alternative to synthetic substances, as they are effective and of 95% ethanol at room temperature for 7 days. The crude
compatible with human and animal life and the environment. extracts were separated by suction filtered through a Bchner
The Piperaceae (pepper) family contains approximately funnel, and the combined filtrates were concentrated to dryness
Vol. 31, no. 1 Journal of Vector Ecology 139

with a rotary evaporator at 60 C until the solvent completely scanning electron microscopic study using low vacuum, the
evaporated. The ethanolic extract of each plant was thus treated larvae were submerged in liquid nitrogen for a few
obtained, lyophilized, and then refrigerated at -20 C until minutes and then attached to aluminum stubs with double-
testing for mosquitocidal activity. stick tape. Ultrastructural details were observed and
photographs taken with SEM (JEOL JSM-5910LV).
Mosquitoes
A laboratory colony of Ae. aegypti mosquitoes, originally Statistical analysis of data
collected in Chiang Mai province, was colonized continuously It was necessary to obtain not less than three mortality
for over 20 generations in a laboratory free of exposure to counts of between 10% and 90%. Experimental tests with
pathogens and insecticides at the Department of Parasitology, more than 20% control mortality were discarded and then
Faculty of Medicine, Chiang Mai University. The adults were repeated. However, when the control mortality ranged from
reared in a room maintained at 25-30 C and 80-90% relative 5-20%, the percentage mortality observed (%M) was
humidity under a photoperiod of 14:10 h (light/dark). In corrected by Abbotts formula (Abbott 1925):
addition to 10% sucrose and 10% multivitamin syrup (SEVEN
SEAS ), adult females were periodically blood-fed on %M = % test mortality -% control mortality x 100
restrained rats to obtain protein for egg maturation. Finely 100-% control mortality
ground dog biscuit was used as a food source for the larvae.
Under these conditions, the life cycle of this mosquito from Dosage-mortality lines were estimated by a computerized
egg to adult took about 3-4 weeks. Early 4th instar larvae were log-probit analysis (Harvard Programming; Hg1, 2). The 95%
used in the experiments. confidence intervals (CI) at the lethal dosage of 50% and 95%
(LD50 and LD95) were used to measure differences between
Larvicidal bioassay test samples.
The test for the larvicidal effect of ethanolic extract
derived from Piper spp. against Ae. aegypti was conducted RESULTS AND DISCUSSION
in accordance with the WHO standard method (WHO 1981).
Each pepper extract was dissolved in absolute ethanol or Ethanolic extractions of the three Piper species, P.
dimethylsulphoxide (DMSO) to prepare a graded series of longum, P. ribesoides, and P. sarmentosum, provided semi-
concentrations. Batches of 25 early 4th instar larvae of Ae. solid materials with various strengths of odor and average
aegypti were transferred in 25 ml of distilled water to a 500 yields of 8.89, 3.21, and 5.30% (w/w), respectively (Table
ml enamel bowl containing 224 ml of distilled water and 1.0 1). In this study, early 4th instar larvae of Ae. aegypti, under
ml of a serial dilution of each plant extract. Four replicate laboratory conditions, were subjected to a dose-dependent
tests were carried out simultaneously, with a final total of 100 efficacy of the three Piper extracts (Table 2). Following
larvae for each concentration. The toxicity of each plant extract treatment of increasing dosages of P. longum, P. sarmentosum,
was evaluated with four to six concentrations yielding a range and P. ribesoides from 1.5-3.5, 2-10, and 6-12 ppm,
of 0-100% mortality. Controls received ethanol- or DMSO- respectively, the larval mortality increased from 20.3-96.3,
distilled water, while the untreated larvae were maintained in 12.3-97.7, and 16.3-90.0%, respectively. No pupal or adult
distilled water only. After treatment, symptoms in treated emergence was observed in the treatment, as complete
larvae were observed and recorded immediately and at time- mortality occurred. In the case of the control or untreated
intervals, and no food was offered to the larvae. The larvae group, mortality observed within 24 h was zero and the larvae
were considered dead if, at the end of 24 h, they showed no developed into pupae and then adults within 48-72 h. Among
sign of swimming movements even after gentle touching with the extracts, P. longum showed more remarkable larvicidal
a glass rod, as described in the World Health Organizations potential than P. sarmentosum and P. ribesoides, presenting
technical report series. The dead larvae in four replicates were LC50 and LC95 values of 2.23 and 4.80, 4.06 and 12.06, and
combined and expressed as a percentage mortality of each 8.13 and 14.01 ppm, respectively. Variety of types and levels
concentration. Bioassays were performed at 25-30 C, of active constituents in each Piper species may be responsible
replicated three times with mosquitoes from different rearing for the variability in their potential against Ae. aegypti.
batches, and reported for the average of individual test results. Symptomatological observations on the larvae treated
with the three Piper extracts revealed a similar manner of
Light and scanning electron microscopy toxicity in the larvae. All larvae were still active immediately
After treatment with a lethal dosage (LC99) of each pepper after exposure to LC99 of each pepper extract, and the feeding
extract, the dead larvae were studied for morphological process and normal zigzag motion of these treated larvae were
alterations under light and scanning electron microscopy. clearly seen. However, after 5 min of exposure, abnormal
Larvae mounted with Hoyers medium on a microscope slide evidence of excitation, restlessness, and sluggishness was
were scrutinized under light microscopy. Morphological initially observed. Excitation and restlessness persisted for
changes in body segments including the head, thorax, and between 10-30 min, and other anomalous motions were seen
abdomen, and other organs such as the eyes, antennae, such as a coiling movement. The treated larvae frequently
mouthbrushes, setae, saddle, and anal gills were observed, sank down and floated up again quickly. During the period of
photographed, and compared with those of the controls. For 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 than those observed in the plant-treated larvae.
bowl. Afterwards, more and more larvae exhibited the toxic Observations on the morphological alterations of treated
symptoms. Appearance of excitation, restlessness, tremors, 4th instar larvae revealed that most organs, except anal papillae
and convulsions followed by paralysis was clearly seen. Two (gills), had a normal structural appearance. Under light and
h after treatment, more than one-half of the larvae were scanning electron microscopes, both treated and control larvae
paralyzed and had sunk to the bottom of the bowl. Moribund showed similarities in morphological architecture and
or dead larvae were increasingly found from 2 to 7 h. At the cuticular sculpturing of the head, thorax, and abdomen
end of a 24-h exposure period, all larvae had subsequently segments, and other organs such as the eyes, antennae,
died. The apparent results indicated a delayed type of larval mouthbrushes, setae, saddle, siphon, and ventral brushes. A
killing from these Piper species. Our findings corresponded distinct difference, however, was the structural alteration of
to those of earlier studies (Insun et al. 1999, Dharmagadda et the anal gills observed in the pepper-treated larvae. This organ
al. 2005, Choochote et al. 2004, 2005), which were carried is involved in the regulation of electrolyte levels. Under light
out to evaluate the larvicidal potential of plant-derived microscopy (Figures 1 and 2), the internal structures of the
materials against some mosquito species. Although symptoms anal papillae in treated larvae showed remarkable shrinkage,
of larvae exposed to the plant oils or extracts, Kaempferia while the external features were normal in appearance.
galanga, Tagetes patula, Apium graveolens, Curcuma Ultrastructural studies (Figures 3 and 4), however, clearly
aromatica, and Piper species seem to be similar, the times to demonstrated external destruction, with extensive damage and
their showing toxic symptoms were relatively different. a shrunken cuticle of the anal papillae. These results therefore
Similarity in the behavior of larvae treated with these plants indicated that the toxic effect of pepper extract is
to those of nerve poisons, i.e. excitation, convulsions, predominantly on the anal papillae, leading to their
paralysis, and death, indicated that the plants probably had a morphological deformation. These findings corresponded to
toxic effect on the neuromuscular system (Sakthivadivel and those of earlier works that investigated the effect of plant-
Thilagavathy 2003, Choochote et al. 2004). Nevertheless, the derived compounds on some species of mosquitoes. Studies
symptoms caused by nerve poisons happened more rapidly conducted by Insun et al. (1999) revealed the severely
142 Journal of Vector Ecology June 2006

morphological disruption of anal papillae observed in dead et al. (2002) reported an effect of larvicidal constituents
Cx. quinquefasciatus larvae. After treatment with ethanolic isolated from P. nigrum fruit against the three mosquito
extract of K. galanga, damaged anal papillae, with a shrunken species, Cx. pipiens pallens, Ae. aegypti, and Ae. togoi.
cuticle border and destroyed surface with loss of ridge-like Larvicidal activity against Ae. aegypti was more marked in
reticulum were found under light and scanning electron retrofractamide A (0.039 ppm) than quineensine (0.89 ppm),
microscopes, respectively. Green et al. (1991) reported distinct pellitorine (0.92 ppm), and pipercide (0.1 ppm). In the study
features of alteration such as highly swollen anal papillae of of Yang et al. (2002), one active isolated from the hexane
Ae. aegypti larvae after treatment with whole oil of Tagetes fraction of the P. longum fruit, pipernonaline, showed potent
minuta. Structural deformation of anal papillae probably led larvicidal activity against Ae. aegypti with LC50 of 0.25 mg/l.
to their dysfunction, which may be intrinsically associated These promising Piper-derived larvicides are results of the
with the death of mosquito larvae. search for new phytochemical agents from Piperaceae plants,
The two pairs of anal papillae are flexible, sac-like which should influence further research of other plants
structures consisting of an epithelium covered by cuticle and belonging to this family in order to find affordable natural
situated on an extension of the terminal segment of mosquito substances for use in mosquito control. Although the larvicidal
larvae. In the fresh-water mosquito larvae, uptake and potential of crude ethanolic extracts derived from the three
elimination of most ions occur via the anal papillae, while the Piper species observed in this study was significantly lower
process of ion conservation is mainly located in the alimentary than that in previous works, further studies for the isolation
canal (Garrett and Bradley 1984, Clements 1992). The and identification of bioactive compounds would be useful
capacity to take up sodium, potassium, chloride, and in developing new types of mosquito larvicides.
phosphate ions from the medium was markedly reduced or Investigations on the toxic effect and mode of action of
lost in papilla-less larvae (Koch 1938, Hassett and Jenkins bioactive compounds against mosquito larvae were previously
1951, Ramsay 1953). This indicated that the lack or carried out. Utrastructural observations on Ae. aegypti larvae
dysfunction of the anal papillae probably led to an interruption after treating with ethanolic extract of Derris urucu roots
of the osmotic and ionic regulation. Although the lower limits revealed an imperfect peritrophic matrix and extensive
of ion concentrations that permit survival of mosquito larvae damage of the midgut epithelium (Gusmo et al. 2002).
have not yet been established, ionic imbalance resulting from Correspondingly, the toxic effect of ethanolic-extracted
the interruption of ionic regulation is also a harmful condition. Magonia pubescens on Ae. aegypti larvae was mainly in the
Considerable research on the larvicidal potential of midgut, showing partial or total cell destruction, high
natural products for controlling Aedes mosquitoes has been citoplasmatic vacuolization, increased subperitrophic space
carried out, but with varied results. Essential oils extracted and cell hypertrophy, and the epithelium did not maintain its
from nine plants commonly found in northeastern Brazil monolayer appearance (Arruda et al. 2003). Generally,
exhibited various degrees of larvicidal activity against Ae. insecticides enter insects by cuticular penetration and/or
aegypti, with LC50 values that ranged from 60 to 538 ppm ingestion and then pass throughout the interior of the insect
(Cavalcanti et al. 2004). Ethanolic extracts from fruit to the site of action (Matsumura 1975). Although these reports
endocarps of Melia azedarach and Azadirachta indica, two provided some evidence regarding the action site of these
members of the family Meliaceae, were found to have lethal bioactive compounds, the mechanism causing mortality of
effects on Ae. aegypti larvae, with LC50 values ranging from mosquito larvae is still unknown and needs to be studied
0.017 to 0.034 g% (Wandscheer et al. 2004). The effective further. However, this study demonstrated and emphasized
constituents in leaf essential oils of Cinnamomum the potential of Piperaceae plants against Ae. aegypti larvae
osmophloeum Provenances including cinnamaldehyde, and its benefit to developing new types of larvicides used for
eugenol, anethole, and cinnamyl acetate showed an inhibitory mosquito control.
effect against the 4th instar larvae of Ae. aegypti, with LC50
values of lower than 50 ppm (Cheng et al. 2004). The LC50 of Acknowledgments
cinnamaldehyde, which had the strongest larvicidal activity,
was 29 ppm. Larvicidal determination of seventeen Brazilian The authors acknowledge the staff members of the
plants demonstrated that five oils derived from Anacardium Department of Parasitology, Faculty of Medicine, Chiang Mai
occidentalis, Copaifera langsdorffii, Carapa guianensis, University for their cooperation. We thank Budsabong
Cymbopogon winterianus, and Ageratum conyzoides, and one Kuntalue and the staff of the Electron Microscopy Research
ethanolic extract of Annona glabra showed high activities and Service Center (EMRSC), Chiang Mai University for their
against Ae. aegypti larvae, with LC50 values of 14.5, 41, 57, technical assistance. Acknowledgment is extended to the
98, 148, and 27 mg/l, respectively (de Mendona et al. 2005). Faculty of Medicine for its financial support of this research
Comparison with the results mentioned above reveals that project and to the Faculty of Medicine Endowment Fund for
the larvicidal potential of Piper spp. tested in this study was Research Publication for its financial support in publishing
greater than or comparable to that of previously described this paper.
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
Vol. 31, no. 1 Journal of Vector Ecology 143

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