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Antimosquito activity of aqueous kernel extract of soapnut Sapindus emarginatus: Impact on various developmental stages of three vector mosquito species and nontarget aquatic insects

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  • Sir Theagaraya College Chennai

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Aqueous (physiological saline) extracts of seed kernel from seven indigenous plants were initially screened for their antimosquito activity against eggs, larvae of all instars, and pupae of Aedes aegypti. Among various seed kernels tested, the soapnut Sapindus emarginatus (Sapindaceae) extract was found to exhibit, for the first time, a strong antimosquito activity as evident from its ability to inflict 100% mortality of all the developmental stages of A. aegypti. Furthermore, the kernel extract of S. emarginatus also exerted ovicidal, larvicidal, and pupicidal activity against two other important vector mosquitoes, namely, Anopheles stephensi and Culex quinquefasciatus. Differential susceptibility of the various developmental stages of the three mosquito species exposed to soapnut extract was also noticed. The kernel extract was found to be safe for two nontarget aquatic insects tested: the larvae of Chironomus costatus and the nymphs of Diplonychus rusticus. Lethal concentration values of soapnut extract to these nontarget insects were always threefold or fivefold higher than those that produced 100% mortality of the larvae of the three mosquito species examined. The findings of this study clearly demonstrate that the aqueous kernel extract of S. emarginatus has potent antimosquito activity detectable against all the developmental stages of three important vector mosquito species as well as it is safe for nontarget aquatic organisms, and thus this new botanical resource could be used as an eco-friendly alternative biocidal agent in control of mosquitoes.
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ORIGINAL PAPER
Antimosquito activity of aqueous kernel extract of soapnut
Sapindus emarginatus: impact on various developmental
stages of three vector mosquito species and nontarget
aquatic insects
Arunagirinathan Koodalingam &
Periasamy Mullainadhan &Munusamy Arumugam
Received: 28 June 2009 / Accepted: 15 July 2009 / Published online: 5 August 2009
#Springer-Verlag 2009
Abstract Aqueous (physiological saline) extracts of seed
kernel from seven indigenous plants were initially screened
for their antimosquito activity against eggs, larvae of all
instars, and pupae of Aedes aegypti. Among various seed
kernels tested, the soapnut Sapindus emarginatus (Sapin-
daceae) extract was found to exhibit, for the first time, a
strong antimosquito activity as evident from its ability to
inflict 100% mortality of all the developmental stages of A.
aegypti. Furthermore, the kernel extract of S.emarginatus
also exerted ovicidal, larvicidal, and pupicidal activity
against two other important vector mosquitoes, namely,
Anopheles stephensi and Culex quinquefasciatus. Differen-
tial susceptibility of the various developmental stages of the
three mosquito species exposed to soapnut extract was also
noticed. The kernel extract was found to be safe for two
nontarget aquatic insects tested: the larvae of Chironomus
costatus and the nymphs of Diplonychus rusticus. Lethal
concentration values of soapnut extract to these nontarget
insects were always threefold or fivefold higher than those
that produced 100% mortality of the larvae of the three
mosquito species examined. The findings of this study
clearly demonstrate that the aqueous kernel extract of S.
emarginatus has potent antimosquito activity detectable
against all the developmental stages of three important
vector mosquito species as well as it is safe for nontarget
aquatic organisms, and thus this new botanical resource
could be used as an eco-friendly alternative biocidal agent
in control of mosquitoes.
Introduction
Vector mosquitoes are capable of transmitting potential
pathogens to human beings, and they are responsible for
several infectious diseases like malaria, filariasis, Japanese
encephalitis, yellow fever, dengue, and chikungunya
(Nauen 2007). They have, therefore, become a challenging
problem to public health worldwide, and it has a serious
social and economical impact especially in tropical and
subtropical countries (Borovsky 2003; Spielman 2003;
Bossche and Coetzer 2008). Mosquito-borne diseases are
endemic over 100 countries, causing mortality of nearly
two million people every year, and at least one million
children die of such diseases each year, leaving as many as
2,100 million people at risk around the world (Kundsen and
Slooff 1992; Weir and Stewart 1997; Klempner et al. 2007).
In India, various species of Aedes,Anopheles, and Culex
mosquitoes are important insect vectors of human diseases,
and the recent outbreak of chikungunya, whose causative
viral agent spread by Aedes aegypti, has affected more than
1.25 million people in 151 districts within eight states of
this tropical country (Pialoux et al. 2007).
Rapid increase in human population, allocation of
limited funds for mosquito control program, and lack of
awareness among people together with environmental
change and adaptability of vector mosquitoes resulted in
an increase in mosquito-transmitted diseases (Gubler 1998).
Thus, the effort towards mosquito control continues to be
an important strategy in preventing the mosquito-borne
diseases (Billingsley et al. 2008). Use of synthetic
chemicals with insecticidal properties such as organo-
chlorines, organophosphates, carbamates, and pyrethroids
has been proven to be the most important effective method
to control mosquitoes and other insect pests all over the
A. Koodalingam :P. Mullainadhan (*):M. Arumugam
Laboratory of Pathobiology, Department of Zoology,
University of Madras,
Guindy Campus,
Chennai 600 025, India
e-mail: mnadhan@yahoo.com
Parasitol Res (2009) 105:14251434
DOI 10.1007/s00436-009-1574-y
world. Nevertheless, their extensive and indiscriminate
applications fostered not only several environmental and
health concerns but also widespread development of
resistance by mosquitoes (Roberts and Andre 1994; Nauen
2007) and unwarranted toxic or lethal effects on nontarget
organisms (Milam et al. 2000). Due to such undesirable
features of chemical insecticides, several investigators have
resorted to explore plant resources to find alternate and eco-
friendly compounds with potent antimosquito activity
(Murugan and Jeyabalan 1999; Chansang et al. 2005; Amer
and Mehlhorn 2006a).
Plants synthesize a variety of primary and secondary
metabolites with insecticidal properties (Carlini and Grossi-
de-Sá 2002; Shaalan et al. 2005). Over 340 plants were
already screened for their antimosquito properties (Sukumar
et al. 1991; Shaalan et al. 2005). Conventionally, the active
ingredients of the plants were extracted mainly with organic
solvents but less frequently attempted with aqueous media
(Sagar and Sehgal 1996; Sivagnaname and Kalyanasundaram
2004;Parketal.2005; Shaalan et al. 2005; Chowdhury et al.
2008; Ghosh et al. 2008; Rahuman et al. 2009). However, the
method of extract preparation with organic solvents as
well as the use of organic solvent extracts is relatively
difficult for routine applications at community level.
Alternatively, aqueous extraction of the desired active
component(s) from the botanical sources could be consid-
ered ideal since such extraction procedure is relatively
simple as well as it allows for easy adoption by the local
communities. Besides, very little effort has been made by
the earlier investigators to test the impact of botanical
biocides on nontarget organisms, and even such limited
studies were mostly performed using active components
extractable with nonpolar solvents (Shaalan et al. 2005).
As an exception, neem-based insecticides mostly extracted
with nonpolar solvents were tested against a large number
of nontarget aquatic and terrestrial organisms (Rao et al.
1995;Kreutzweiser1997; Kreutzweiser et al. 2000;Boeke
et al. 2004).
Among many well-known phytochemicals, azadirachtin
(a tetranotriterpenoid compound) is one of the most exten-
sively studied biological insecticides extracted from the seed
kernel of the neem tree Azadirachta indica (Schmutterer
1990). This secondary metabolite is known to be effective
against a wide range of insect pests as well as vectors
(Su and Mulla 1999; Rong et al. 2000; Huang et al. 2004;
Sriwattanarungsee et al. 2008), and its efficacy against the
mosquito targets has been proven by its ovicidal (Su and
Mulla 1999), larvicidal, and pupicidal activity (Coria et al.
2008) and inhibition of insect growth (Okumu et al. 2007).
Despite these merits, there are, however, at least two notable
disadvantages with azadirachtin: it degrades rapidly upon
exposure to sunlight, resulting in great loss of its insecticidal
activity (Johnson et al. 2003), and azadirachtin (0.5 and
1 ppm) in water suspension enhances the oviposition response
of the mosquito Culex tarsalis, and this enhancement was
more pronounced in 1-week-old neem formulation (Su and
Mulla 1999). Such limitations observed with the well-known
botanical biocide necessitate exploration of new alternative
biocidal compound(s) from other plant sources with potent
antimosquito activity.
Indeed, aqueous extracts from numerous plant materials
have been screened for their antimosquito property by
earlier investigators, but these studies are, however,
incomplete in the following aspects: (1) the aqueous
extracts were often tested for their toxicity on one or two
mosquito species, (2) only arbitrarily selected developmen-
tal stages of mosquito have been used, and (3) the impact of
aqueous extract with potent mosquitocidal property was not
extensively examined against nontarget organisms. It is,
therefore, desirable to test empirically the impact of
aqueous extracts of various plants on eggs (ovicidal), larvae
of all instars (larvicidal), and pupae (pupicidal) of major
vector mosquitoes in order to explore a single botanical
biocide with potent mosquitocidal property against various
mosquito species. This contention is valuable since various
developmental stages of the mosquitoes are inhabiting
aquatic medium, and it is, therefore, relatively easy to deal
with them within this confined habitat (Amer and Mehlhorn
2006a).
In this study, we have screened aqueous extracts of
diverse indigenous plants to explore a new source of plant
material with potent antimosquito activity against various
developmental stages of the three major vector mosquito
species as well as examined the impact of an empirically
chosen aqueous plant extract on nontarget organisms.
Methods
Mosquito rearing
Eggs of A.aegypti were collected within the university
campus by placing water-filled plastic trays (23 × 15 ×
6.5 cm) with a lining of partially immersed filter paper.
The egg rafts of Culex quinquefasciatus and the larvae of
Anopheles stephensi were collected from Adyar river near
Saidapet, Chennai, and transferred to cups containing
unchlorinated tap water and then brought to the laboratory.
The eggs were placed in enamel trays (30× 24×5 cm) each
containing 2 l of tap water and kept at room temperature
(26±2°C) with a photoperiod of 16:8 h (L:D) for larval
hatching. The larvae of each mosquito species were
maintained in separate trays under the same laboratory
conditions and fed with a powdered feed containing a
mixture of dog biscuit and baker's yeast (3:1 ratio). The
trays with pupae of each mosquito species were maintained
1426 Parasitol Res (2009) 105:14251434
in separate mosquito cages at 26 ± 2°C and relative humidity
of 85±3% under a photoperiod of 16:8 h (L:D) for adult
emergence. Cotton soaked in 10% aqueous sucrose solution
in a petri dish to feed adult mosquitoes was also placed in
each mosquito cage. An immobilized young chick was
placed for 3 h inside the cage in order to provide blood
meal especially for female mosquitoes. A plastic tray (11×
10×4 cm) filled with tap water with a lining of partially
immersed filter paper was then placed inside each cage to
enable the female mosquitoes to lay their eggs. The eggs
obtained from the laboratory-reared mosquitoes were
immediately used for toxicity assays or allowed to hatch
out under the controlled laboratory conditions described
above. Only the newly hatched specific instars of larvae or
the pupae of different mosquito species were used in all
bioassays.
Maintenance of nontarget insects
The larvae of Chironomus costatus (Chironomidae: Dip-
tera) were collected from Adyar river near Thiruverkkadu,
Chennai, and brought to the laboratory in the water taken
from the collection site. Among various larval instars, only
the fourth-instar larvae, identified by their head capsule
width and body length, were separately collected and used
for bioassays.
Only adult male water bugs, Diplonychus rusticus
(Belostomatidae: Hemiptera) carrying eggs on their elytra,
were collected from the freshwater ponds in Chetpet,
Chennai, and they were brought to the laboratory in the
water taken from the collection site. The water bugs were
maintained in the laboratory in plastic trays (30 × 25 ×6 cm)
containing tap water at room temperature (26± 2°C) in the
prevailing normal photoperiod (16:8 h/L:D). The bugs were
fed ad libitum with the larvae of C.quinquefasciatus. The
first-instar nymphs which hatched out from the eggs were
collected and immediately used for bioassays.
Plant materials
The seeds of Adenanthera pavonia and Pongamia glabra
were collected from our university campus, and the fruits of
Artocarpus integrifolia,Pithecellobium dulce, and Sapin-
dus emarginatus were purchased from the local market. The
pods of Bauhinia variegata and Canavalia ensiformis were
collected from the areas around the agricultural fields
located near Kovalam and Uthukkottai (suburban places
near Chennai), respectively. The plant materials were
identified by Prof. R. Balasubramanian (Center for Ad-
vanced Studies in Botany, University of Madras). The seed
coat of the dried seeds from the seven plants was manually
removed, and the kernels alone were used for preparation of
various extracts.
Preparation of aqueous extracts
The dried seed kernels of each plant were fine-powdered
using an electric mixergrinder for 5 min. The kernel
powder (20 g) from each plant source was added to 60 ml
of physiological saline (0.9% NaCl solution), mixed well,
and then made up to 100 ml with saline (20% w/v). All
samples were kept overnight at 10°C and then stirred well
on a magnetic stirrer for 3 h at 25°C. The resulting slurry
were sonicated (20 kHz/100 W; 4×30 s, 2°C) in Labsonic L
ultrasonicator using a standard probe 5 T (B. Braun,
Germany). Each kernel extract was centrifuged (8,000×g,
30 min, 4°C); the resulting supernatant was filtered using
Whatman no. 1 filter paper; the clear filtrate was stored at
10°C and used within a week.
Estimation of dry weight
The dry weight of the extract was determined gravimetri-
cally using a sample (1 ml) with known weight of each
extract, which was completely dried in a desiccator
containing fused calcium chloride. The dry weight was
determined for each freshly prepared extract for successive
bioassays. Although 20 g of kernels from each plant source
was used to prepare the aqueous extract, the dry weight per
milliliter of supernatant obtained from each type of kernels
considerably varied, apparently due to variation in the
amount of water-soluble biochemical components present
in kernels from different plant species.
Laboratory bioassays
The biocidal activity of the test materials were evaluated in
the laboratory essentially following the guidelines recom-
mended by the World Health Organization (1996). Five
different volumes (ranging from 1 to 10 ml) of each extract
were diluted separately to 50 ml with unchlorinated tap
water (test media) to obtain various concentrations (1 to
12 mg dry weight per milliliter) of the extracts, which were
subsequently used for exposure of various developmental
stages of the mosquito. The final effective or lethal
concentration of each extract preparation in the test medium
was always reported in milligram dry weight per milliliter.
Ovicidal assay
The ovicidal assay was performed by placing batches of ten
mosquito eggs in 50 ml of each test medium in a plastic
bowl containing a specific concentration of the aqueous
kernel extract to be tested. In control, the same number of
eggs was maintained in 50 ml of unchlorinated tap water
containing appropriate volume of 0.9% saline. All contain-
ers were maintained at room temperature (26 ± 2°C) with
Parasitol Res (2009) 105:14251434 1427
naturally prevailing photoperiod (18:6 h/L:D) in the
laboratory. The test medium was replaced after 48 h with
fresh medium containing the same extract at the testing
concentration. Water lost through evaporation was com-
pensated by periodic addition of unchlorinated tap water.
All the test media were carefully examined every 24 h up to
96 h for the number of intact (unhatched) eggs as well as
the appearance of the number of first-instar larvae, and the
latter indicated the successful egg hatchability. This
maximum time point for egg hatchability was fixed since
the embryogenesis in mosquitoes under normal condition
has been reported to be completed within 96 h (Judson and
Gojrati 1967). Besides, the unhatched eggs remaining in the
test media after 96 h of exposure were transferred to tap
water and maintained up to 24 h in order to ascertain the
mortality of these eggs. The eggs that failed to hatch out
even under this ideal condition were considered to be dead
due to their previous exposure to a particular test medium.
Percentage mortality of the eggs, representing the ovicidal
effect of the test material, was calculated from the total
number of eggs introduced into the medium and the number
of unhatched or dead eggs.
Larvicidal/pupicidal assay
Batches of ten larvae of each instar or pupae were
introduced into 50 ml of the test medium (tap water) in a
plastic bowl containing particular concentration of the
aqueous kernel extract. In control, the same number of
larvae at a particular instar or pupae was maintained in
50 ml of unchlorinated tap water containing appropriate
volume of 0.9% saline. All containers were maintained at
room temperature (26±2°C) with naturally prevailing
photoperiod (18:6 h/L:D) in the laboratory. The larvae
were fed ad libitum with a mixture of dog biscuit and
baker's yeast (3:1 ratio). Water lost through evaporation was
compensated by periodic addition of unchlorinated tap
water. Any larva or pupa was considered to be dead if its
appendages did not move when prodded repeatedly with a
wooden dowel. Mortality of each larval instar or pupae was
recorded after 24 and 48 h of exposure to the extract.
Impact on nontarget aquatic insects
Batches of ten fourth-instar larvae of C.costatus or five
first-instar nymphs of D.rusticus were separately intro-
duced into 50 ml of test medium in plastic bowls containing
various concentrations (2, 4, 6, 8, and 10 mg dry weight per
milliliter) of aqueous kernel extract of the soapnut. Controls
received appropriate volumes of 0.9% saline in 50 ml of tap
water. The larvae of Chironomus and the nymphs of the
water bug were fed ad libitum with a mixture of dog biscuit
and baker's yeast (3:1 ratio) and live larvae of C.
quinquefasciatus, respectively. Water lost through evapora-
tion was compensated by periodic addition of unchlorinated
tap water. The test organisms were periodically observed
for their swimming activity and behavioral responses
during the experimental period. Mortality of the test
organisms were recorded after 24 and 48 h of exposure.
Determination of lethal concentrations
Lethal concentration (LC
50
) represents the concentration of
the test material that caused 50% mortality of the test (target
and nontarget) organisms within the specified period of
exposure, and it was determined by exposing various
developmental stages of the mosquitoes to different
concentrations of the extract. Based on the mortality of
the test organisms recorded in these bioassays, LC
50
was
calculated along with their fiducial limits at 95% confi-
dence level by probit analysis using SPSS software package
(version 10.0). It is notable that the eggs, larvae of all
instars, pupae of different mosquito species, and two
nontarget aquatic insects maintained in appropriate control
media survived well without any incidence of mortality up
to 48 h or 96 h as in the case of mosquito eggs.
Results
In the screening study, the aqueous extract of seed kernels
from seven different plants were tested for their lethal
effects on various developmental stages (eggs, larvae of all
instars, and pupae) of the mosquito A.aegypti. The aqueous
extract of seed kernels from diverse plant sources caused
mortality of various developmental stages of A.aegypti
with varying degrees (Table 1). Among the extracts of seed
kernels from seven different plants tested, the extract
obtained from three seed types, namely, P.glabra,A.
pavonia,andS.emarginatus, were found to exhibit
relatively more effective ovicidal, larvicidal, and pupicidal
activity (60100% mortality at 48 h), whereas the seed
kernel extracts of P.dulce inflicted the lowest mortality of
various developmental stages of A.aegypti. Of the three
effective seed kernel extracts, only the extract of S.
emarginatus (soapnut) was found to kill (100% mortality)
all the developmental stages of the mosquito within 48 h at
a concentration of as little as 5 mg dry weight per milliliter
as compared with the effective concentrations of kernel
extracts from two other plants examined. Based on these
results, a detailed investigation was subsequently focused
only on the most effective seed kernel of S.emarginatus.
The lethal effect of S.emarginatus was further examined
using various concentrations of seed kernel extract on eggs,
larvae of all four instars, and pupae of A.aegypti (Tables 2
and 3). The aqueous kernel extract at varying concen-
1428 Parasitol Res (2009) 105:14251434
trations caused 100% mortality of all the developmental
stages of the mosquito tested. The minimal concentrations
of this extract that caused a maximal (100%) mortality of
the eggs, larvae of all the four instars, and pupae were 2.5
(96-h exposure), 2.5 to 4.0 (24-h exposure), and 4.5 mg
(24-h exposure) dry weight per milliliter, respectively.
Furthermore, LC
50
(96-h exposure) for the ovicidal effect
of the aqueous kernel extract was 1.12 mg dry weight per
milliliter, and relatively higher extract concentrations were
required to produce similar effects within 24 h against
various stages of larval instars (LC
50
1.51 to 2.57 mg dry
weight per milliliter) and pupae (LC
50
3.52 mg dry weight
per milliliter). This extract was effective even at a lesser
concentration especially against the larvae and pupae of the
mosquito when exposure time was extended to 48 h (LC
50
1.31 to 2.43 and 3.12 mg dry weight per milliliter,
respectively).
As presented in Tables 2and 4, the aqueous kernel extract
of the soapnut also caused 100% mortality of all the
developmental stages of mosquito A.stephensi at the effective
concentrations of 2.5 for eggs (96 h), 2.5 to 4.0 for first- to
fourth-instar larvae (24 h), and 4.5 mg dry weight per
milliliter for pupae (24 h). The LC
50
values determined for
the corresponding developmental stages were 1.08, 1.64 to
3.34, and 3.64 mg dry weight per milliliter. This extract at
relatively lower concentrations effectively caused mortality of
various larval instars and pupae of A.stephensi upon
extending their exposure period to 48 h (LC
50
1.47 to 3.12
and 3.47 mg dry weight per milliliter, respectively).
Similarly, the aqueous extract of S.emarginatus kernels
exhibited potent ovicidal, larvicidal, and pupicidal property,
producing 100% mortality of another species of mosquito,
C.quinquefasciatus, at the concentration of 2.5 mg dry weight
per milliliter for eggs (96 h), 2.5 to 4.0 mg dry weight per
milliliter for the first to fourth larval instars (24 h), and 4.5 mg
dry weight per milliliter for the pupae (24 h). The corres-
ponding LC
50
valueswere1.29,1.80to3.42,and4.13mgdry
weight per milliliter. Upon extension of the exposure period to
48 h, all the four larval instars and pupae of the mosquito
became susceptible to lethal effect of the extract relatively at
lesser concentrations (LC
50
1.59 to 3.13 and 3.91 mg dry
weight per milliliter, respectively; Tables 2and 5).
The effect of S.emarginatus extract against two
nontarget aquatic insects, namely, C.costatus and D.
rusticus, is presented in Table 6. This extract did not cause
mortality of fourth-instar larvae of C.costatus at a
concentration of 2 mg dry weight per milliliter up to 48 h,
whereas no mortality of the first-instar nymphs of the
aquatic bug D.rusticus was observed up to a concentration
of 6 mg dry weight per milliliter within 48 h of exposure. In
both cases, the larvae and nymphs were active without any
perceptible change in their normal behavior. The larvae of
Table 1 Mosquitocidal property of aqueous seed kernel extracts from a variety of plants tested against various developmental stages of Aedes
aegypti
Plant materials screened Maximum tested concentration
(mg dry weight/ml)
a
Mortality (%) of various developmental stages of mosquito tested (48h)
b
Eggs Larval instars Pupae
I II III IV
Pithecellobium dulce 11 35 43 35 20 15 20
Bauhinia variegata 10 55 70 35 27 20 20
Artocarpus integrifolia 10 53 100 83 37 27 30
Canavalia ensiformis 11 67 100 100 84 80 90
Pongamia glabra 10 100 100 100 70 60 70
Adenanthera pavonia 12 100 100 100 100 100 94
Sapindus emarginatus 5 100 100 100 100 100 100
a
Final concentrations in the test media
b
Data represent mean values from five determinations for each plant material using samples from different preparations
Table 2 Antimosquito activity of aqueous kernel extract of soapnut
(S.emarginatus) against various developmental stages (eggs, larvae,
and pupae) of three mosquito species, namely, A.aegypti,A.
stephensi, and C.quinquefasciatus
Developmental stages tested Minimal lethally effective
concentration (mg dry weight/ml)
a
24h 48h
Eggs (96-h exposure) 2.5
First-instar larvae 2.5 2.5
Second-instar larvae 3.0 2.5
Third-instar larvae 4.0 3.5
Fourth-instar larvae 4.0 4.0
Pupae 4.5 4.5
a
Final concentrations in the test media at which 100% mortality was
recorded
Parasitol Res (2009) 105:14251434 1429
C.costatus exposed to higher concentrations of this extract
showed mortality at or above 4 mg dry weight per milliliter,
reaching 100% mortality at a concentration of 8 mg dry
weight per milliliter (24-h exposure) with the LC
50
value of
5.71 and 4.78 mg dry weight per milliliter for 24 and 48 h,
respectively. The mortality (35%) of the nymphs of D.
rusticus was first recorded upon their exposure to the
extract concentration of 8 mg dry weight per milliliter for
48 h, and 100% mortality of the nymph was observed only
when the nymphs were exposed for the same period to the
extract concentration of 10 mg dry weight per milliliter,
with the LC
50
values of 9.29 and 8.26 mg dry weight per
milliliter for 24- and 48-h exposure time, respectively.
In a comparative analysis (Fig. 1), LC
50
(24 h) values
recorded for the two nontarget aquatic insects (C.costatus
and D.rusticus) exposed to various concentrations of
soapnut kernel extract were found to be always at least
threefold or fivefold higher (5.71 and 9.29 mg dry weight
per milliliter, respectively) than the mean LC
50
(24 h) value
obtained for the first-instar larvae (1.65 mg dry weight per
milliliter) of all the three mosquito species tested.
Discussion
Plants make up a vast repository for primary and secondary
metabolites with a wide range of biological activity
including antimosquito property. In this connection, it is
notable that extensive screening studies performed by Amer
and Mehlhorn (2006a) have demonstrated the potent
larvicidal effects of essential oils from 13 out of 41 plants
tested, in which the best oils inflicted 100% mortality
within about 24 h of exposure of the larvae of A. aegypti,A.
stephensi, and C.quinquefasciatus. Besides, some of these
oil preparations have been reported to induce 100%
repellency for 8 h against all the three adult mosquito
species (Amer and Mehlhorn 2006b). A number of earlier
investigators have also examined the impact of aqueous
extracts (distilled water or tap water) of different parts of a
variety of plants on various or selected developmental
stages of one or two species of mosquito (Murugan and
Jeyabalan 1999; Chansang et al. 2005; Khanna and
Kannabiran 2007). Among such studies, Chansang et al.
(2005) screened nine different plants and reported that the
Table 3 Antimosquito activity of aqueous kernel extract of soapnut (S.emarginatus) against various developmental stages (eggs, larvae, and
pupae) of mosquito A.aegypti
Developmental stages tested 24-h exposure 48-h exposure
Lethal
concentration
a
(mg dry weight/ml)
95% lower and upper
confidence limits
(mg dry weight/ml)
Lethal
concentration
a
(mg dry weight/ml)
95% lower and upper
confidence limits
(mg dry weight/ml)
Eggs (96-h exposure) LC
50
1.12 0.131.46
First-instar larvae LC
50
1.51 1.291.84 LC
50
1.31 0.921.58
Second-instar larvae LC
50
2.02 1.762.31 LC
50
1.72 1.451.97
Third-instar larvae LC
50
2.50 1.992.82 LC
50
2.29 1.922.54
Fourth-instar larvae LC
50
2.57 2.252.84 LC
50
2.43 2.022.71
Pupae LC
50
3.52 3.103.82 LC
50
3.12 2.103.47
a
Lethal concentration was calculated using mean values from eight determinations using samples from different preparations
Table 4 Antimosquito activity of aqueous kernel extract of soapnut (S.emarginatus) against various developmental stages (eggs, larvae, and
pupae) of mosquito A.stephensi
Developmental stages tested 24-h exposure 48-h exposure
Lethal concentration
a
(mg dry weight/ml)
95% lower and upper
confidence limits
(mg dry weight/ml)
Lethal concentration
a
(mg dry weight/ml)
95% lower and upper
confidence limits
(mg dry weight/ml)
Eggs (96-h exposure) LC
50
1.08 0.281.34
First-instar larvae LC
50
1.64 1.371.90 LC
50
1.47 1.171.72
Second-instar larvae LC
50
1.85 1.592.12 LC
50
1.65 1.371.91
Third-instar larvae LC
50
2.38 2.062.85 LC
50
1.98 1.692.28
Fourth-instar larvae LC
50
3.34 2.933.60 LC
50
3.12 2.843.48
Pupae LC
50
3.64 3.353.91 LC
50
3.47 3.133.73
a
Lethal concentration was calculated using mean values from eight determinations using samples from different preparations
1430 Parasitol Res (2009) 105:14251434
aqueous extract of the fruits from Piper retrofractum
showed the highest larvicidal effect on the third- and
fourth-instar larvae of A.aegypti and C.quinquefasciatus.
Another similar screening study performed by Khanna and
Kannabiran (2007) using three different plants has
demonstrated the highest lethal effect of aqueous root
extract of Hemidesmus indicus on the larvae of C.
quinquefasciatus. A few earlier investigators have also
attempted to extract the bioactive compounds with anti-
mosquito activity using both polar and nonpolar solvents
and compared the efficiency of these two extraction
methods. Phukan and Kalita (2005) attempted to extract
the bioactive compound with antimosquito activity from
the leaf powder of Litsea salicifolia using polar (water)
and various nonpolar solvents (hexane, toluene, chloro-
form, acetone, and methanol) among which the aqueous
extract, as compared with other organic solvent extracts,
exhibited more effective larvicidal activity against the
fourth-instar larvae of A.aegypti. By contrast, Chowdhury
et al. (2008) extracted the bioactive compounds from
Solanum villosum berryusingdistilledwaterandfive
different organic solvents and detected the higher mortal-
Table 5 Antimosquito activity of aqueous kernel extract of soapnut (S.emarginatus) against various developmental stages (eggs, larvae, and
pupae) of mosquito C.quinquefasciatus
Developmental stages tested 24-h exposure 48-h exposure
Lethal concentration
a
(mg dry weight/ml)
95% lower and upper
confidence limits
(mg dry weight/ml)
Lethal concentration
a
(mg dry weight/ml)
95% lower and upper
confidence limits
(mg dry weight/ml)
Eggs (96-h exposure) LC
50
1.29 0.831.56
First-instar larvae LC
50
1.80 1.472.09 LC
50
1.59 1.271.85
Second-instar larvae LC
50
2.14 1.872.45 LC
50
2.01 1.742.29
Third-instar larvae LC
50
2.81 2.523.07 LC
50
2.53 2.322.76
Fourth-instar larvae LC
50
3.42 2.903.92 LC
50
3.13 2.563.63
Pupae LC
50
4.13 3.504.67 LC
50
3.91 3.194.45
a
Lethal concentration was calculated using mean values from eight determinations using samples from different preparations
Table 6 Analyses of lethal effects of aqueous kernel extract of soapnut (S.emarginatus) on two nontarget aquatic insects: fourth-instar larvae of
C.costatus and first-instar nymphs of the aquatic bug D.rusticus
Mortality (%)a
Lethal concentrationb
(mg dry weight / ml)
Non-target insects
tested
No. of larvae /
nymphs used
per treatment
Tested concentrations
(mg dry weight / ml) 24 h 48 h 24 h 48 h
2 0 ± 0 0 ± 0
4 21 ± 6 30 ± 9
6 40 ± 7 81 ± 9
8 100 ± 0 100 ± 0
Chironomus
costatus
(fourth instar
larvae)
10
10 100 ± 0 100 ± 0
LC50 5.71
LC50 4.78
2 0 ± 0 0 ± 0
4 0 ± 0 0 ± 0
6 0 ± 0 0 ± 0
8 35 ± 10 35 ± 10
Diplonychus
rusticus
(first instar
nymphs)
5
10 65 ± 10 100 ± 0
LC50 9.29
LC50 8.26
a
Data represent mean ± SD of five determinations using samples from different preparations
b
Lethal concentration was calculated using mean values from five determinations using samples from different preparations
Parasitol Res (2009) 105:14251434 1431
ity against the third-instar larvae of A.aegypti with all
organic solvent extracts than the aqueous extract.
In the present study, the aqueous (physiological saline)
extract of kernels from the soapnut S.emarginatus was
found to exhibit, for the first time, potent antimosquito
activity as evident from its stronger ovicidal, larvicidal, and
pupicidal effects by inflicting 100% mortality of all these
developmental stages of A.aegypti within 24 h of exposure.
An attempt was also made in this study to extract the active
component(s) with antimosquito activity from the soapnut
kernel powder using double-distilled water, physiological
saline, and four different organic solvents (acetone, meth-
anol, chloroform, and petroleum ether) with varying polarity
in order to gain an insight into the tentative biochemical
identity of the active component(s). In the bioassays
performed with the second-instar larvae of A.aegypti,the
methanol extract exerted the highest larvicidal effect as
compared with the three other organic solvent extracts
tested, and the efficacy was closely followed by physiolog-
ical saline and distilled water extract (data not shown). It is,
therefore, apparent that the compound(s) with antimosquito
activity present in the kernel is extractable with polar as
well as nonpolar solvents, thereby suggesting that the active
component(s) responsible for the antimosquito activity
detected in the soapnut kernel is not associated with a
singular type of biochemical compound. However, the
aqueous extract was preferably used in further investigations
for its following features: (1) effective extraction of the
phytochemicals with antimosquito activity, (2) this solvent is
eco-friendly, and (3) the extraction procedure is relatively
simple that could be eventually adopted by local community.
In a subsequent study, the aqueous extract of soapnut
was found to exhibit 100% mortality of eggs (within 96 h),
all the four larval instars, and pupae (24 h) of the three
mosquito species tested at the effective concentrations of
2.5 to 4.5 mg dry weight per milliliter. In these bioassays,
the individuals at the earlier developmental stages were
invariably more vulnerable to this extract, a finding in
accordance with a few earlier studies performed with
selected developmental stages of the mosquitoes (Redwane
et al. 2002; Cetin et al. 2004; Lapcharoen et al. 2005;
Nathan et al. 2006; Chowdhury et al. 2008). Besides, the
toxicity of the S.emarginatus extract was not only
sustained in the aquatic medium but also enhanced upon
extension of the exposure period up to 48 h. Comparative
analysis of LC
50
values obtained for each developmental
stage of the three mosquito species tested in this investiga-
tion revealed their differential susceptibility to the soapnut
extract. Accordingly, the eggs, second- and third-instar
larvae of A.stephensi, and first- and fourth-instar larvae as
well as pupae of A.aegypti were more susceptible to the
extract. By contrast, all the developmental stages of the C.
quinquefasciatus were relatively less susceptible than the
two other mosquito species tested. In a similar study,
Sivagnaname and Kalyanasundaram (2004) reported that A.
aegypti was more susceptible to the leaf extract of Atlantia
monophylla followed by C.quinquefasciatus and A.
stephensi. Lapcharoen et al. (2005) also demonstrated that
A.aegypti was more susceptible than C.quinquefasciatus
when exposed to three plant extracts. The differences in the
susceptibility of different mosquito species to the plant
extracts observed in this study may be attributed to inherent
differences in physiological mechanisms among various
species of mosquitoes tested.
Kreutzweiser (1997) reported the deleterious effects of
neem extract and a commercial neem formulation (Azatin)
on a variety of eight species of nontarget aquatic inverte-
brates with the highest lethal effect on the larvae of mayfly
(Isonychia bicolor/rufa). In a similar study, Sivagnaname
and Kalyanasundaram (2004) have tested the toxicity of A.
monophylla extract on five nontarget mosquito predators
and demonstrated that this extract was highly toxic to the
back-swimming hemipteran water bug, Anisops bouvieri,
and moderately toxic to another water bug Diplonychus
indicus (D.rusticus). The potent antimosquito activity
detected in our study with the aqueous extract of soapnut
kernel against all the developmental stages of three vector
mosquito species gave an impetus to test its impact on two
nontarget aquatic animals, namely, C.costatus and D.
rusticus. The soapnut extract at LC
50
dose for the first-
instar larvae of the three mosquito species failed to produce
any impact on two nontarget insects tested, and this extract,
however, tends to produce mortality of these two nontarget
insect species only upon their exposure to higher extract
concentrations. It is also notable from these bioassays that
the lethal concentrations (LC
50
for 24 h) of the aqueous
extract of the soapnut kernel determined for both the
nontarget insects tested were always threefold or fivefold
higher than those actually required for any of the three
10
9
8
7
6
5
4
3
2
1
0
LC50 (mg dry weight / ml)
Mosquito*C. costatus D. rusticus
*The value depicted represents mean LC50 value obtained
for the first instar larvae of three species of mosquitoes tested.
Fig. 1 Comparison of LC
50
(24 h) values of soapnut S.emarginatus
kernel aqueous extract for the two nontarget aquatic insects (larvae of
C.costatus and nymphs of D.rusticus) and the larvae of target
mosquitoes
1432 Parasitol Res (2009) 105:14251434
mosquito species examined in this study. Although this
short-term experimental study was restricted to two
nontarget aquatic animals, the results from the bioassays,
nevertheless, revealed that the lethal concentrations of the
soapnut kernel extract for the mosquito larvae were well
below the lethal thresholds for the nontargets tested. All
these observations suggest that this extract is apparently
safe for aquatic nontarget organisms, and the prime action
of the biocidal component(s) in the extract appears to be
more towards various developmental stages of the mosqui-
toes. Thus, the findings of this study clearly demonstrate
the potent antimosquito property of aqueous extract of
kernels from the soapnut S.emarginatus as evident from its
ability to kill all the developmental stages of three impor-
tant vector mosquito species, and it also appears to be safe
for the nontarget organisms. Further studies are needed to
evaluate the identity of the bioactive component(s) of this
extract and its systemic effects on target mosquitoes, which
would eventually enable the application of the soapnut
extract as an eco-friendly biocidal agent for the effective
control of vector mosquitoes.
Acknowledgements This work was supported by a grant (University
with Potential for Excellence) from the University Grants Commission,
New Delhi, under the Herbal Science Research Program through the
University of Madras to P.M. (HS-29). We thank Dr. S. Janarthanan for
his critical reading of the manuscript and members of the Laboratory of
Pathobiology for their support and encouragement.
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... Sapindus emarginatus, another tree from this genus, widely distributed in India, has also demonstrated larvicidal activity of its fruit extract against three important vector mosquitoes: A. aegypti, Anopheles stephensi and (Diptera: Culicidae) (Koodalingam et al., 2009). Later, this group has also investigated the impact of the extracts on the activity of mosquito phosphatases and esterases to gain an insight into the extent of disturbance in metabolic homeostasis inflicted upon exposure to the extract (Koodalingam et al., 2011). ...
... Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) (Porras & Lopez-Avila, 2009); and complete ethanolic extracts from fruits have shown larvicidal and morphological alterations effects on the mosquito Aedes aegypti (Diptera: Culicidae) (Ferreira Barreto et al., 2006). Some other saponins presenting other kinds of biological activity, isolated from the fruits of this species, are shown in Figure 1 (Lemos et al., 1992; Ribeiro et al., 1995).Sapindus emarginatus, another tree from this genus, widely distributed in India, has also demonstrated larvicidal activity of its fruit extract against three important vector mosquitoes: A. aegypti, Anopheles stephensi and Culex quimquefasciatus (Diptera: Culicidae)(Koodalingam et al., 2009). Later, this group has also investigated the impact of the extracts on the activity of mosquito phosphatases and esterases to gain an insight into the extent of disturbance in metabolic homeostasis inflicted upon exposure to the extract(Koodalingam et al., 2011). ...
... Later studies were also conducted which resulted that exposure of the kernel extract produces changes in total proteins, and esterases, phosphatases resulting in metabolic disturbances. The extract shows its anti-mosquito activity by multiple modes of action as clear from several adverse changes noticed in three main enzymes, namely, Acetylcholinestrase, βcarboxyl esterase and acid phosphatases of larvae of A. aegypti [21,22]. ...
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Soapnuts and other saponin-rich plant materials are known for their phytochemistry and pharmacology. After a gap of two decades, there has been a sudden spate in research on soapnuts with experiments on a wide range of applications. The present review compiles these different aspects of research to explore the possibility of creating a circular economy around soapnut. We are looking at the study from the cradle-to-grave approach. Of the twelve soapnut species present globally, this paper focuses on three- Sapindus mukorossi, S. trifoliatus syn laurifolia, and S. emarginatus. The saponin content varies among the three species, making it difficult to ascertain the critical micelle concentration (CMC), an important functional aspect. Recent research in applications includes surfactants in industry, laundry, bioremediation, biopesticide, poultry feed supplement, biodiesel, biochar, and pharmacology. As an alternative to laundry detergent, soapnut works best at CMC. The ecological services of the tree are restricted to terrestrial ecosystems, while the fruit is toxic to aquatic animals. More research is needed to establish the permissible limits for soapnut saponins in wastewater and their biodegradability before soapnuts can be accepted as a bio-based surfactant. Nevertheless, research indicates that it will be beneficial to propagate soapnuts as a sustainable supplement to petroleum-based surfactants and fuels. With many by-products from soapnuts, it is possible to attain zero wastage. Propagation techniques, including natural regeneration, selective crop breeding, vegetative propagation, and tissue culture, have been explored to promote high-quality crops. Planting the appropriate variety of soapnuts could provide a sustainable agroforestry crop that is resilient to climate change.
Article
This work deals with the evaluation of nutritional and medicinal potential of a defatted kernel of Sapindus mukorossis seed. Defatted sapindus seed kernel is a rich source of proteins (33.4 ± 2.12%), which show balanced amino acid composition for the requirement of adults as per the World Health Organization. Protein isolate possesses 29 kDa molecular weight peptide, which shows trypsin inhibitor activity. It also showed around 31.2% reduction in amylase activity while aqueous Ethanol and ethanol extracts showed 55% and 72.83%, respectively. Aqueous ethanol and ethanol extracts were found to contain polyphenols and saponins. Polyphenol content in aqueous ethanol and ethanol extract was 4.50 ± 0.15 mg/g and 5.7 ± 0.34 mg/g ferulic acid equivalent, respectively, while 0.72 ± 0.68% and 1.2 ± 0.23% Oleonolic acid equivalent saponins, respectively. Both these extracts showed potent antioxidant activity, and the rate of DPPH scavenging activity was higher than the ferulic acid standard. The deffated seed also contains dietary fibers in which 3.8 ± 0.01% are soluble, and 2.2 ± 0.03% are insoluble fibers.
Article
ABSTRACT The Anti anxiety activity of Ethanolic extract of Sapindusemarginatus flowers was determined by experimental animal models like Elevated plus maze, Despair swim test and Haloperidol induced catalepsy.The plant was collected in the month of March from Erode city and was extracted by Macceration using ethanol as solvent. The anxiolytic activity of the plant was studied using Diazepam (0.5mg/kg, i.p) as standard and with two different doses of extract (200mg/kg & 400mg/kg, Oral) by using elevated plus maze, despaire swim test and haloperidol induced catalepsy. The time spent by the test group in the open arm was found significant when compared to the standard in Elevated plus maze with both the doses of extract. The immobility time (reaction time) was significantly reduced in the test group when to compare to that of the standard in Despair swim test. The animals in the test group had a failure to correct an externally imposed unusual posture over a prolonged period of time in Catalepsy.The above studies have been concluded that the ethanolic extract of Sapindusemarginatus flowers exhibit anxiolytic effect in experimental rats. So it supports the use of Sapindusemarginatusflowers as anxiolytic agents. Further investigations should be made to elucidate the active constituent of responsible for the activity.
Article
Vector-borne diseases are an increasing cause of death and suffering worldwide. Efforts to control these diseases have been focused on the use of chemical pesticides, but arthropod resistance (whether physiological, biochemical, or behavioral) to pesticides is now an immense practical problem. The pharmacokinetic interactions of pesticides with arthropods, mechanisms of resistance, and the strengths and shortcomings of different resistance test methods are briefly reviewed. Using malaria control as an example, the differences between the efficacy of insecticide-sprayed houses in reducing malaria transmission, and the actual effect of such treatments on vectors are discussed. Reduced malaria transmission as a result of spraying house walls occurs through some combination of killing vectors that land on sprayed walls (insecticidal effect) and by preventing vectors from entering or remaining inside long enough to bite (behavioral effects). Both insecticidal and behavioral effects of insecticides are important, but the relative importance of one versus the other is controversial. Field studies in Africa, India, Brazil, and Mexico provide persuasive evidence for strong behavioral avoidance of DDT by the primary vector species. This avoidance behavior, exhibited when malaria vectors avoid insecticides by not entering or by rapidly exiting sprayed houses, should raise serious questions about the overall value of current physiological and biochemical resistance tests. The continued efficacy of DDT in Africa, India, Brazil, and Mexico, where 69% of all reported cases of malaria occur and where vectors are physiologically resistant to DDT (excluding Brazil), serves as one indicator that repellency is very important in preventing indoor transmission of malaria. This experience with DDT has implications for future control efforts because pyrethroids also stimulate avoidance behaviors in arthropods. Each chemical should be studied early (before broad-scale use) to define types of action against vector species by geographic area, especially for impregnated bed net applications. The problems for vector control created by use of insecticides in agriculture and the potential for management of resistance in both agriculture and vector-borne disease control are discussed.
Article
Azadirachtin-A when exposed to UV light (254 nm), as a solid thin film on a glass surface, furnished only a single photoproduct. The photoproduct was isolated by repeated column chromatography and identified by NMR and mass spectroscopy. NMR data indicated that the (E)-2-methylbut-2-enoate ester group of azadirachtin-A has been converted into (Z)-2-methylbut-2-enoate ester. Half-life of azadirachtin-A as thin film under UV light was found to be 48 min.
Article
Recent requirements for biomonitoring of urban stormwater runoff have raised the issue of toxic contributions from mosquito control products. A comparison of seven pesticides for their toxicity to target and nontarget organisms was conducted in field and laboratory trials to determine relative impacts in and around Craighead County, Arkansas. Twenty-four and forty-eight-hour acute toxicity tests using Ceriodaphnia dubia, Daphnia magna, Daphnia pulex, and Pimephales promelas were employed with U.S. Environmental Protection Agency (U.S. EPA) suggested procedures as standard test organisms. Additional tests with resident mosquito fish, Gambusia affinis, and mosquito larvae, Anopheles quadrimaculatus, included ditch-receiving waters to compare the somewhat sterile laboratory exposures to actual field conditions. Exposure to as much as 31.4 μg/L of the pesticides Dursban®, malathion, Permanone®, Abate®, Scourge®, B.t.i, and Biomist® were required for effective control of An. quadrimaculatus, whereas as little as 2.7 μg/L resulted in substantial mortality of some nontarget organisms. These data suggest that prevailing application rates for effective mosquito control not only affect nontarget organisms but may also confound stormwater and nonpoint toxicity evaluations that utilize sensitive indicator species.
Article
Outdoors sream channels weret teated with a commercial neem formulation, Neemixx® 4.5, and a neem extract (no formulation ingredients) to determine the effects on aquatic insect communities. An exposure period of 5 h was chosen to simulate the transient nature of pesticide residues in streams and rivers after aerial applications. Multivariate analytic procedures based on Bray-Curtis similarity matrices were used to compare community structure among the replicate channels 9 d after treatment. No significant differences in community structure were found among controls and channels treated with Neemix at an azadirachtin concentration of 0.28 mg/L, but a multivariate measure of community stress indicated an increase in variability among treated channels. Significant differences in community structure were found among controls and channels treated with Neemix at 0.84 and 2.54 mg/L, and this resulted from reductions in several key taxa. During subsequent experiments with a neem powder extract, the formulation ingredients of Neemix were at least partially responsible for the significant effects on community structure at 0.84 mg/L azadirachtin. No significant differences were found among controls and channels treated with the extract at 0.9 mg/L, whereas the community structure of aquatic insects in channels treated at 3.0 mg/L differed significantly from controls. In a Canadian forest pest-management context, the expected environmental concentration in water bodies of areas sprayed with azadirachtin at 50 g/ha is 0.035 mg/L.
Article
Neem (Azadirachta indica A. Juss) products have been shown to exert pesticidal properties against a variety of insect species. In mosquito control programs, such products may have the potential to be used successfully as larvicides. In exploring other advantages of neem products, we studied the oviposition responses of Culex tarsalis Coquillett and Cx.quinquefasciatus Say to two experimental azadirachtin (AZ) formulations, wettable powder Azad – WP10 (WP) and emulsifiable concentrate Azad – EC4.5 (EC). Gravid Cx.tarsalis exhibited a distinct preference for the neem suspension of the WP, where significantly more egg rafts were collected from the treatment than from the control. The minimum effective AZ concentration for this activity was 0.5ppm. The aged suspensions from 1–7days at 0.5 and 1ppm AZ were more active in eliciting oviposition responses in Cx.tarsalis than the fresh preparations. This activity of the aged suspensions lasted up to 14 and 21days at 0.5 and 1ppm AZ, respectively. Negative ovipositional responses were indicated in the tests of the EC vs. Cx.tarsalis, as well as both neem formulations vs. Cx.quinquefasciatus. In the tests of the EC formulation, significantly less gravid females were trapped by oviposition cups in the treatment than in the control, and in the tests of the WP significantly less egg rafts were collected from the treatment than from the control. The minimum effective concentrations for oviposition avoidance activity were 5ppm AZ for Cx.tarsalis and 10ppm AZ for Cx.quinquefasciatus, which lasted up to 1 and 4 days for these two species respectively. Neem products potentially used as mosquito larvicides may have many additional benefits in mosquito control programs, the oviposition modification noted in the current studies is one such example.
Article
Eggs of Aedes aegypti (L.) were submerged in water containing dissolved oxygen at levels ranging from less than 1 to 14 parts per million, at 1, 24, 48, 72 and 96 hours after being laid. After a 4.5 day exposure period, which encompasses the normal period of embryogeny, the eggs were subjected to the hatching stimulus as a measure of maturity. The whole of embryogeny occurred at a normal rate under levels of 3.8 to 14 ppm dissolved oxygen. An oxygen level of 0.95 ppm was lethal to all eggs except those exposed only in the advanced stages of development. A level of 1.9 ppm dissolved oxygen caused a retardation of developmental rate, with 6.5 days being required to achieve maturation. Immature, but advanced, embryos could be ‘hatched’ artificially, with completion of development to normal adults. DIE WIRKUNGEN UNTERSCHIEDLICHER SAUERSTOFFSPANNUNGEN AUF DIE EMBRYOGENESE UND LARVALREAKTIONEN VON AEDES AEGYPTI Eier von Aedes aegypti wurden 1, 24, 48, 72 und 96 Stunden nach der Ablage in Wasser getaucht, das gelösten Sauerstoff in Mengen von weniger als 1 bis 14 Teilen pro Million enthielt. Nach einer Behandlungszeit von 4, 5 Tagen, die dem normalen Zeitraum der Embryonalentwicklung entspricht, wurden die Eier als Maß ihrer Reife dem Schlüpfreiz unter-worfen. Die gesamte Embryonalentwicklung verlief bei Sauerstoffspannungen von 3, 8 bis 14 ppm in normalem Ausmaß. Eine Sauerstoffspannung von 0, 95 ppm war für alle Eier lethal mit Ausnahme derjenigen, die ihr erst in fortgeschrittenen Entwicklungsstadien ausgesetzt wurden. Eine Menge von 1, 9 ppm gelösten Sauerstoffs verursachte eine Verzögerung der Entwicklungs-geschwindigkeit, bei der 6, 5 Tage zur Erreichung der Schlüpfreife benötigt wurden. Unreife, aber fortgeschrittene Embryonen konnten künstlich zum Schlüpfen gebracht werden, bei vollständiger Weiterentwicklung zu normalen Imagines.
Article
Azadirachtin, as a botanical insecticide, affects a wide variety of biological processes, including reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using proteomic techniques, we analyzed changes in protein metabolism of Spodoptera litura (F.) induced by azadirachtin. Following feeding 4th instar larvae of Spodoptera litura (F.) with an artificial diet containing 1 ppm azadirachtin until pupation, 48 h old pupae were collected and protein samples prepared. Total soluble protein content was measured and the results showed that azadirachtin significantly influenced protein level. Moreover, the proteins were separated by 2-DE (two-dimensional polyacrylamide gel electrophoresis) and 10 proteins were significantly affected by azadirachtin treatment when compared to an untreated control. Six of these proteins were identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. These proteins are involved in various cellular functions. One identified protein may function as an ecdysone receptor, which regulates insect development, and reproduction. It is suggested that the botanical insecticide azadirachtin affects protein expression and the azadirachtin-related proteins would be essential for a better understanding of the mechanisms by which neem toxins exert their effects on insects.
Article
Aqueous extract obtained from deoiled neem and karanja seed kernels (ADNSD and ADKSK) were assessed for their toxic and growth regulating activities against Cx quinquefaciatus treated as first instar larvae. ADNSK at various concentrations was effective on the growth regulating mechanism, inducing prolonged larval stages. However, 100% larval mortality was observed, especially during the first and the second instars at all the tested concentrations. ADKSK caused 100% mortality in the fourth instar larvae and prepupae at the concentration of 100 ppm with no significant effect on the developmental period. The adults emerging from treated (50 ppm) larvae were smaller in size and malformed. We found ADNSK to be more effective than ADKSK.