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DOI 10.1007/s00436-013-3528-7
ORIGINAL PAPER
activity against mosquito vectors (Amer and Mehlhorn lined with Whatman filter paper on all the sides and half-filled
2006a, b; Rahuman et al. 2008a, b; Kumar et al. 2012a), with dechlorinated tap water. The eggs were allowed to hatch
though the insecticidal effects of plant chemicals vary not in trays filled with dechlorinated water. Larvae were fed upon
only according to plant species, mosquito species, and plant a mixture of finely ground dog biscuits and yeast in the ratio of
parts, but also with extraction methodology. Phytochemicals 3:1 by weight. Care was taken to prevent formation of any
do not pose any threat to the environment and also possess scum on the surface of water. The pupae formed were collect-
multiple effects against vector mosquitoes which include ed and transferred to the cloth cages for adult emergence.
growth regulation, fecundity suppression, male sterility, lar-
vicidal, ovicidal, and oviposition activity mostly as deter- Plant collection
rence (Kumar et al. 2010, 2011a, b). Nevertheless, the mos-
quito control potential of weed plants found in vast areas on Different parts of the A. mexicana plant, i.e., leaves, stems,
plains as well as on hilly regions has not been attempted at and roots, were collected from the surrounding areas in New
large scale (Raj Mohan and Ramaswamy 2007; Kumar et al. Delhi, India. The collected parts were thoroughly washed
2012b). Only a few reports are available regarding the po- with tap water and dried under the shade at room temperature
tential of certain weeds as larvicidal, ovicidal, and oviposi- of 272 C separately for about 20 days. The dried parts
tion deterrent agent against A. aegypti (Sathish and were then crushed, powdered, and sieved thoroughly to get
Maneemegalai 2008; Kumar et al. 2011a, 2012b). fine powder.
Argemone mexicana is a common weed which is used in
the traditional system of medicine to cure malarial fever and Preparation of the extract
ulcers. Its seeds are efficient in treating leprosy, dropsy, and
jaundice (Alagesaboopathi 2009). Certain bioactive com- The powdered plant parts, i.e., roots, stems, and leaves, were
pounds derived from A. mexicana seeds have been proven weighed separately. Of each powdered material, 200 g was
to possess chemosterilant activity including reduction in extracted in 1,000 mL of petroleum ether, hexane, benzene,
blood meal utilization, diminished fecundity, formation of acetone, and ethanol, separately using Soxhlet extraction
larvalpupal or pupaladult intermediates, adult mortality, and apparatus. The extraction continued for 3 days, 8 h per day,
sterility of first-generation eggs in A. aegypti (Sakthivadivel at a temperature not exceeding the boiling point of the
and Thilagavathy 2003). The available literature, however, solvent. The crude extracts, thus formed, were concentrated
reveals that this plant has not been explored extensively using a vacuum evaporator at 45 C under low pressure.
against mosquito vectors as larvicidal agent or by affecting After complete evaporation of the solvent, the concentrated
their biological characteristics. Hence, this study was under- extracts were collected and stored in a refrigerator at 4 C as
taken to assess the probable use of extracts prepared from the stock solution of 1,000 ppm for further use. This stock
different parts of A. mexicana against dengue vector larvae solution was used to prepare the desired concentrations of
causing mortality, behavioral changes, or morphological alter- the extracts for investigating the cidal, behavioral, and mor-
ations. The assessment of mosquito control potential of this phological effects against larvae of A. aegypti.
weed, besides its management may be useful in promoting
research aiming at the development of new mosquito control Screening of extracts for their larvicidal efficacy against A.
agent. aegypti
Table 1 Screening of different extracts (1,000 ppm) prepared from Statistical analysis of data
leaf, stem and root of A. mexicana for larvicidal activity against dengue
vector A. aegypti
The larvicidal tests with more than 20 % mortality in controls
S. no. Part used Extract Mortality after 24 h (%) and pupae formed were discarded and repeated again. If the
control mortality ranged between 5 and 20 %, it was
1 Leaf Hexane extract 100
corrected using Abbotts formula (Abbott 1925).
Petroleum ether extract 100
The data were subjected to regression analysis using com-
Benzene extract 57.5
puterized SPSS 16.0 Program. The LC50 and LC90 values with
Acetone extract 50
95 % fiducial limits were calculated in each bioassay to
Ethanol extract 70
measure difference between the test samples.
2 Stem Hexane extract 100
Petroleum ether extract 100 Behavioral studies in extract-treated A. aegypti larvae
Benzene extract 75
Acetone extract 60 During each larvicidal bioassay, the larvae were monitored
Ethanol extract 57.5 carefully for behavioral modifications, if any, caused by
3 Root Hexane extract 100 extract-mediated disruption of biological functions. The be-
Petroleum ether extract 100 havioral observations included wriggling speed, horizontal
Benzene extract 77.5 movements, vertical movements, aggregation behavior, and
Acetone extract 15 knockdown. The larval behavior was recorded and
Ethanol extract 25 photographed with Canon Power Shot SX50HS. Similar
observations were made in controls for comparison with
treated larvae.
The dead and moribund larvae were recorded after 24 h. Morphological studies in extract-treated A. aegypti larvae
Similar tests were carried out with each extract against early
fourth instars of A. aegypti to assess the larvicidal efficiency After treatment, the dead larvae were scrutinized for mor-
of A. mexicana. phological alterations under light microscopy. Morphologi-
cal modifications in body segments including the head, tho-
Evaluation of larvicidal potential of selected extracts rax, and abdomen, and other organs such as the eyes, anten-
against A. aegypti nae, mouth brushes, setae, saddle, and anal gills were ob-
served, photographed, and compared with those of the con-
The extracts that failed to give 80100 % larval mortality at trols. The larvae were also observed carefully for any
1,000 ppm were considered ineffective and not tested further changes in pigmentation pattern.
for larvicidal efficacy. Other extracts causing 80100 % larval
mortality at 1,000 ppm were evaluated further for larvicidal
potential. The bioassays were performed as described earlier. Results
Four replicates were carried out simultaneously for each assay
making a total of 80 larvae for each concentration of each The potential of leaf, stem, and root extracts of A. mexicana in
extract. Controls were exposed to the solvent, i.e., ethanol five different solvents was studied against A. aegypti larvae for
alone. The larval mortality was recorded after 24 as well as use as ecofriendly insecticides as an alternative for eco-enemy
48 h of exposure. synthetic insecticides. The results of the larvicidal tests
Table 2 Larvicidal activity of the root extracts of A. mexicana against early fourth instars of A. aegypti
Table 3 Larvicidal activity of the leaf extracts of A. mexicana against early fourth instars of A. aegypti
performed against fourth instars of A. aegypti with 1,000 ppm 158.839 ppm after 24 h of exposure, the petroleum ether root
of different extracts prepared in five solvents, i.e., petroleum extract exhibited 54.6 and 88.5 % increased efficacy as com-
ether, hexane, benzene, acetone, and ethanol are presented in pared to the petroleum ether stem and root extracts, respec-
Table 1. The data clearly reveals the varied potential of ex- tively. It is also worthy to note that the toxicity potential
tracts against A. aegypti with only petroleum ether and hexane increased after prolonged periods of exposure of the larvae
extracts as effective larvicides leading to 100 % larval mor- to the extracts; the LC50 values decreasing dramatically after
tality at 1,000 ppm (Table 1). The remaining extracts proved 48 h of exposure with hexane root extract still being the most
to be 2285 % less efficient exhibiting a range of 1578 % effective larvicide (Fig. 2).
larval mortality. The least effective extract was found to be the Behavioral observations on the larvae treated with all the
acetone root extract whereas ethanol leaf extract and benzene effective extracts of A. mexicana revealed interesting behav-
root extract exhibited moderate toxicity (Table 1). The other ioral symptoms. On immediate exposure to the extracts, all
extracts, except hexane and petroleum ether extracts, were not the larvae exhibited a normal feeding and zigzag wriggling
considered for further investigations. motion till 510 min of exposure. Thereafter, the larvae
The larvicidal bioassays carried out with the selected ex- started showing signs of unnatural restlessness and excita-
tracts against A. aegypti revealed that though petroleum ether tion. The wriggling speed increased conspicuously with
extracts were quite effective against dengue vector larvae, aggressive vertical movements rising repeatedly to the water
hexane extracts exhibited 1.1- to 1.8-fold more larvicidal surface. Most interesting observation was the aggressive
potential than the petroleum ether extracts, irrespective of self-biting of anal papillae with their mouth parts leading to
whether these were prepared from roots, stems, or leaves of the formation of ring-shaped structures (Fig. 3). This aggres-
A. mexicana (Tables 2, 3, and 4, Fig. 1). The results further sive pattern persisted for 23 h after which the larvae became
revealed that the extracts prepared from the roots of A. sluggish, failing to reach the water surface, followed by high
mexicana showed 1.6- to 2.4-fold higher efficacy than those levels of larval knockdown due to chronic paralysis. Mori-
prepared from the leaves and stem. It establishes the hexane bund or dead larvae were increasingly found from 4 to 8 h.
root extract to be the most effective larvicide of all the extracts Observations on the morphological alterations of treated
tested resulting in LC50 and LC90 values of 91.331 and larvae revealed that most organs had a normal structural
156.684 ppm, respectively, after 24 of exposure (Table 2). appearance except anal papillae (gills). Under light micro-
The extract proved to be 1.8-fold more toxic than hexane leaf scope, both treated and control larvae showed similarities in
extracts (Table 3) and a 2.4-fold more toxic as against hexane morphological architecture and cuticular sculpturing of the
stem extracts (Table 4). Similarly, with an LC50 value of head, thorax, and abdomen segments, and other organs such
Table 4 Larvicidal activity of the stem extracts of A. mexicana against early fourth instars of A. aegypti
as the eyes, antennae, mouth brushes, setae, saddle, siphon, resistance to synthetic insecticides and the residue problems
and ventral brushes. A distinct difference, however, was the in the environment, the current inclination is to explore plants
structural alteration of the anal gills observed in the extract- for certain phytochemicals that are safe for nontarget animals
treated larvae as compare to the control larvae. The observa- and do not pose any residue problem but are still able to
tions clearly demonstrated destruction of anal papillae with suppress pest populations. During the last decade, various
extensive damage and a shrunken cuticle of the internal mem- studies on natural plant products against mosquito vectors
brane whereas the external membrane was intact (Fig. 4). have renewed the interest in them as possible alternatives to
Another remarkable observation was the demelanization of synthetic chemical insecticides. The botanical extracts from
the cuticle as compared to control (Fig. 5). the plant leaves, roots, seeds, flowers, and bark in their crude
form have been used as conventional insecticides for centu-
ries. In fact, many researchers have reported the effectiveness
Discussion of plant extracts or essential oils against mosquito larvae
(Rahuman et al. 2007; Rawani et al. 2009; Warikoo et al.
Increasing documentation of negative environmental and 2011; Kumar et al. 2012b).
health impact of synthetic insecticides, and growing incidence A. mexicana is an aggressive weed that grows in the tem-
of resistance in the mosquitoes have necessitated the need for perate region in waste lands, cultivating fields, and road sides.
the development of new strategies for mosquito control (Lima In the traditional system of medicine, the whole plant of A.
et al. 2011). Keeping in view the development of insect mexicana is extensively used in the treatment of tumors, warts,
skin diseases, inflammations, rheumatism, jaundice, leprosy, prepared from A. mexicana seeds have been proved to pos-
piles, warm infestations, and dysentery. Along with its pharma- sess larvicidal potential against second instars of dengue
cological properties, a few researchers have reported its poten- vector causing high mortality at test concentrations from 25
tial role in control of mosquito vectors (Sakthivadivel and to 200 ppm (Sakthivadivel and Thilagavathy 2003).
Thilagavathy 2008; Priya and Rao 2012). Keeping in view Our results also confirmed the root extracts as the most
limited research work and antimosquito potential of the plant, effective extracts with 1.6- to 2.4-fold higher efficacies as
the present investigations were carried out to assess the prospec- compared to the leaf and stem extracts. These results are in
tive use of A. mexicana in mosquito management programs. conformity with that of Kumar et al. (2012b) who also report-
Present investigations clearly revealed the efficacy of ed the maximum efficacy of extracts prepared from the roots
extracts prepared from the three parts (leaf, stem, and root) of P. hysterophorus as compared to those prepared from stem
of A. mexicana against fourth instars of A. aegypti; though and leaves against dengue vector larvae. Larvicidal bioassays
only hexane and petroleum ether extracts, out of the five with hexane root extracts of A. mexicana resulted in an LC50
solvents tested, could result in 80100 % mortality. Similar and LC90 value of 91.331 and 156.684 ppm, respectively,
results were reported by Kumar et al. (2012b) who tested against early fourth instars of A. aegypti after 24 h of exposure.
different parts of a weed, Parthenium hysterophorus, The toxicity potential of the extract, however, increased by
extracted in five solvents against A. aegypti larvae and found 1.1-fold on exposure to the extract for 48 h. In comparison,
only hexane and petroleum ether extracts being effective hexane root extracts of P. hysterophorus have been found
causing 100 % mortality. However, the acetone extracts moderately effective against fourth instars of A. aegypti with
to improved formulations with enhanced activity, which may Kumar S, Singh AP, Nair G, Batra S, Seth A, Wahab N, Warikoo R
(2011a) Impact of Parthenium hysterophorus leaf extracts on the
eventually become environmentally acceptable and replace
fecundity, fertility and behavioural response of Aedes aegypti L.
objectionable conventional insecticides for mosquito control. Parasitol Res 108:853859
Kumar S, Warikoo R, Misha M, Seth A, Wahab N (2011b) Larvicidal
Acknowledgments The authors are highly grateful to University Grants efficacy of the Citrus limetta peel extracts against Indian strains of
Commission, New Delhi for providing research fellowship to carry out the Anopheles stephensi Liston and Aedes aegypti L. Parasitol Res
present investigations. Thanks are extended to Dr. Savithri Singh, Principal, 111:173178
Acharya Narendra Dev College for providing infrastructure and research Kumar S, Wahab N, Mishra M, Warikoo R (2012a) Evaluation of 15
facilities. local plant species as larvicidal agents against an Indian strain of
dengue fever mosquito, Aedes aegypti L. (Diptera: Culicidae).
Front Physiol 3:16
Kumar S, Nair G, Singh AP, Batra S, Wahab N, Warikoo R (2012b)
Evaluation of the larvicidal efficiency of stem, roots and leaves of
the weed, Parthenium hysterophorus (Family: Asteraceae) against
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