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International Journal of Research in Botany

Universal Research Publications. All rights reserved

ISSN 23197854
Original Article
Efficacy of Crude extracts of Annona reticulata and Pongamia pinnata as larvicidal for
the Management of filarial vector Culex quinquefasciatus Say Diptera: Culicidae
J. B. Nayak
Department of Botany, Nizam College, Hyderabad, 500 001, Andhra Pradesh, India.
Received 26 December 2013; accepted 02 January 2014
Abstract
Objective: To evaluate the larvicidal activity of crude extracts derived from selected medicinal plants with special
reference to journals and tribal knowledge, against early fourth instar larvae of Culex quinquefasciatus. Say (Diptera:
Culicidae).
Methods: According to WHO guidelines on good agricultural and collection practices (GACP) for medicinal plants the
fresh and healthy plant materials were collected and extracted with methanol and separated by Rotary evaporator. The
efficacy was determined through bioassay method. For bioassay test, twenty numbers of fourth instars larvae were taken in
six batches of twenty each for the treatment. Bowls of 100 ml capacity were kept in series, and tested for each desired plant
extract concentrations 5 to 200 ppm. The control was set up with distilled water. The numbers of dead larvae were counted
after every 12 hrs of treatment up to 48 hours.
Results: These crude extracts have showed 100% mortality within short period. at different concentrations. And no
mortality was found in control.
Conclusions: Present study indicates that crude extracts of Annona reticulata and Pongamia pinnata carry huge potential
as a mosquito larvicidal. This potential could be exploited for the development of safer and effective botanical mosquito
larvicidal tool for the management of Cx. quinquefasciatus.
Keywords: Annona reticulata, Pongamia pinnata, larvicidal activity, Crude extracts, Culex quinquefasciatus.
2013 Universal Research Publications. All rights reserved
1. Introduction notorious insect because in this stage larvae are localized
Mosquitoes are well known as annoying pests and as and restricted to a small space due to the low mobility
carriers of disease-causing agents to humans and animals. (Howard et al., 2007). Moreover, vector control is facing a
Several mosquito species belonging to genera Anopheles, threat due to the emergence of resistance to synthetic
Culex and Aedes are vectors for the pathogens of various insecticides. In this context, essential oils have received
diseases like malaria, filariasis, Japanese encephalitis, much attention as potentially useful bioactive compounds
dengue and dengue hemorrhagic fever, yellow fever, etc. against insects (Mathew and Thoppil, 2011; Massebo et al.,
Culex quinquefasciatus is a vector of lymphatic 2009; Chowdhury et al., 2008).
filariasis, while this disease is widely distributed in tropical The application of easily degradable plant
area with around 120 million people infected worldwide compounds is considered one of the safest methods in
and 44 million people having common chronic controlling insect pests and vectors (Sivagnaname et al.,
manifestation (Bernhard et al ., 2003). Tropical areas are 2004).
more prone to parasitic diseases and the risk has increased Plant extracts are currently studied more and more
due to climate change and intensifying Globalization because of their role in plant protection as well as in urban
(Karunamoorthy et al., 2010). Also, owing to poor drainage entomology (Din et al., 2011). Plant extracts are safer for
system, especially during rainy seasons, the presence of non-target organisms including man, therefore, plant based
many fish ponds, irrigation ditches and the rice fields Formulations would be more feasible from environmental
provide abundant mosquito breeding places. Mosquito perspective than synthetic mosquitocides (Bhat et al.,
vector-borne diseases contribute to the major disease 2009). Herbal products with proven potential as insecticide
burden in India (Prabhu et al., 2011). or repellent can play an important role in the interruption of
Fight against adult mosquitoes is temporary, the transmission of mosquito-borne diseases at the
unsatisfactory, inadequate and environmental polluting. individual as well as at the community level. Aromatic
Larval treatment is much more effective for managing this plants and their essential oils are very important sources of

International Journal of Research in Botany 2014; 4(1): 1-5

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many compounds that are used in different respects (Amer
et al., 2006).
Great deal of research have been done by number
of authors around the globe and significant progress have
been made in the field of management of notorious
mosquito species through the utilization of botanicals
(essential oils/polar and non-polar extracts) derived from
aromatic medicinal plant/spices (Traboulsi et al., 2002;
Prajapati et al., 2005; Lee, 2006; Pitasawata et al., 2007;
Knioa et al., 2008).
2. Materials and methods
2.1. Collection of plant material:
According to WHO guidelines on good
agricultural and collection practices (GACP) for medicinal
plants the fresh and healthy plant materials of Annona
reticulata leaf Fig (a) and Pongamia pinnata leaf Fig (b)
and bark Fig (c) were collected from different places of
Telangana region of Andhra Pradesh, India. In the
individual cleaned mesh bags and plastic bags with the help
of protective clothing gloves, which will prevent and
protect from toxic and dermatitis-causing plants, poisonous
animals and disease-carrying insects. The Medicinal plant
parts were collected With reference to various journals and
local tribal knowledge for the purpose of present study.
Fig (a). Annona reticulata
Fig (b). Pongamia pinnata bark
Fig (c). Pongamia pinnata leaf
International Journal of Research in Botany 2014; 4(1): 1-5
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The collected raw medicinal plant materials like
leaf and bark were transferred to the Lab and immediately
washed with tap water to remove soil, dust and any other
microorganisms, and also to eliminate undesirable
materials and contaminants. The collected medicinal plant
materials were protected from insects, rodents, birds and
other pests, and also from Livestock and domestic animals
Plant species and their parts used for extraction.
Plant Vernacular Plant
Family
species name part
Annona
Rama phalamu Leaf Annonaceae
reticulata
Pongamia Leaf and
pinnata
Kanuga
bark
Fabaceae
2.2. Processing of plant material:
The collected plant material leaf and bark were
shade dried at room temperature for 7- 10 days. The leaves
were shade-dried, and powdered (Maheshwaran et al.,
2008) (Patole and Mahajan 2009). Each sample was
extracted with methanol using a soxhlet apparatus (Vogel,
1978).
The dried plant material was grinded and
powdered mechanically using electrical stainless steel
blender (Model: Sumeet 1.5L Capacity, 6 angle blades, 2
speed w/pulse and made of Stainless Steel Blade. Libra
appliances pvt. Ltd, Baroda, India). The dried and
powdered plant materials were stored in plastic bags. and
each sample was extracted in sample bottles with methanol
by maceration method. After 5 days of the solvent
extraction it was filtered with watman filter paper no.1 and
then solvent extraction was obtained. Next the Solvent was
separated by rotary evaporator (boiling point range 64
65.5 C) for 8 hrs. The pooled methanol extracts were
concentrated separately by rotary vaccum evaporator at 40
C and evaporated to dryness and stored at 4 C in an air
tight bottle (Jang et al., 2002). and the residue obtained was
stored at 4 C in the deep freezer.
2.3. Preparation of stock solution:
One gram of crude extract was first dissolved
in100 ml of methanol and stored as stock solution. The
anthelmintic assay was carried as per the method of Tandon
et al., (1997). This stock solution was used to prepare the
desired concentrations of the extract for the larvicidal
activity on the mosquito larvae.
From the stock solution 5, 10, 25, 50, 100 and 200
ppm concentrations were prepared with dechlorinated tap
water. The control was set up with 100 ml tap water by
adding 2ml of methanol.
3. Selection of Mosquito species:
The important vector species of mosquitoes which
is Culex quinquefasciatus was selected for the present
study. Cx. quinquefasciatus is the vector of west Nile virus
which causes Encephalitis or Meningitis which is known to
affect the brain tissue, finally resulting in permanent
neurological damage (Hubalek and Halouzka, 1999).
3.1 Mosquito larvae collection and culture:
The fourth instar larvae were collected and
transferred in to a glass beaker of 1 liter capacity containing
clean water, and the larvae after sorting were identified as Table 1: Mortality rates of Culex quinquefasciatus mosquito
Cx. quinquefasciatus larvae (Shyamapada Mandal 2011). larvae at different concentrations of crude extract of Annona
Culex quinquefasciatus larvae were collected from reticulata leaves.
Conc. Time (hours) n = 20
stagnant sewage water of the River Musi in Hyderabad. (ppm) 12 24 36 48 Total % Mean SE (+/-)
The identification of mosquito larvae was done by Dr. B. 5 12 15 18 20 20 100 5.000 1.750
Redya Naik, Entomologist, Department of Zoology, 10 17 19 20 - 20 100 6.666 0.764
25 20 - - - 20 100 20.000 0.000
Osmania University, Hyderabad, Andhra Pradesh. The 50 20 - - - 20 100 20.000 0.000
collected larvae were reared from egg to larval stage and 100 20 - - - 20 100 20.000 0.000
then to adults in the laboratory itself, to avoid the species 200 20 - - - 20 100 20.000 0.000
Control 0 0 0 0 0 0 0.000 0.000
mixture. From these adults, next F1 generation larvae were n = number of larvae used for bioassay test
used for the present study. This procedure facilitates to
Graph 1: Mortality rates of Culex quinquefasciatus mosquito
maintain the uniform age of larval instar (fourth instar).
larvae at different concentrations of crude extract of Annona
The larvae were reared in tap water and fed with reticulata leaves.
dog biscuits and yeast tablets at the ratio of 3:1. They were
maintained at 28 2 C, 7585% RH, under 14L: 10D
photoperiod cycles. The larvicidal activity was assessed by
the procedure of WHO (1996). The larvae were fed with
dry yeast powder on the water surface (50mg/1)
(Senthilnathan S. 2007).
4. Bioassay:
The larvicidal activity was assessed by the
standard guidelines of WHO (2005), with some
modification and as per the method of Rahuman et al.,
(2000), Prabakar and Jebanesan (2004). A laboratory
colony of C. quinquefasciatus was used for the larvicidal
activity studies. Table.2. The larvicidal activity of Pongamia
Patole and Mahajan, (2009), reported that, the pinnata bark crude extract at different concentrations is
twenty numbers of 3 to 4th instar larvae were taken out represented in Table: 4. the data shows that, 100 %
from culture through and transferred in to plastic bowls mortality rate was observed at 5 and 10 ppm concentrations
with the help of dropper. of crude extracts after 48 and 24 hrs of the treatment. At the
For bioassay test, twenty numbers of fourth instars concentration of 25, 50, 100 and 200 ppm, 100 % mortality
larvae were taken in six batches of twenty each for the rates were recorded after 12 hrs. The control did not show
treatment. Bowls of 100 ml capacity were kept in series, any mortality. This crude extracts has given excellent
and tested for each desired plant extract concentrations 5, results when compared to that of others.
10, 25, 50, 100 and 200 ppm. The control was set up with 2
ml methanol and distilled water. The experimental media, Table 2: Mortality rates Culex quinquefasciatus mosquito larvae
in which 100% mortality rate of larvae occurred were at different concentrations of crude extract of Pongamia pinnata
bark.
selected for a dose response bioassay. Based on the Conc. Time (hours) n = 20
screening results, crude methanol solvent extracts of leaf (ppm) 12 24 36 48 Total % Mean SE (+/-)
and bark extracts of the plant listed were subjected to dose 5 10 15 18 20 20 100 10.00 2.500
10 19 20 - - 20 100 10.00 0.250
response bioassay for larvicidal activity against the larvae 25 20 - - - 20 100 20.00 0.000
of Cx. quinquefasciatus. The numbers of dead larvae were 50 20 - - - 20 100 20.00 0.000
counted after every 12 hrs of treatment up to 48 hours. The 100 20 - - - 20 100 20.00 0.000
200 20 - - - 20 100 20.00 0.000
percentage mortality and standard error of mean have been Control 0 0 0 0 0 0 0.000 0.000
calculated for all the results obtained by this study. n= number of larvae used for bioassay test.
5. Results and Discussion: Graph 2: Mortality rates of Culex quinquefasciatus mosquito
Table.1. the larvicidal activity of A. reticulata leaf larvae at different concentrations of crude extract of Pongamia
crude extract at different concentration is represented in pinnata bark.
Table: 2. the data shows that, 100 % mortality rate of larvae
was observed at 5, 10, 25, 50, 100 and 200 ppm
concentrations of crude extract. The concentration of 5 ppm
showed 100 % mortality rate was recorded after 48 hrs of
the treatment. And for 10, 25, 50, 100 and 200 ppm it was
observed after 12 hrs of the treatment. The control did not
show any mortality.
The present investigation is the pioneer work on the
Annona reticulata methanolic leaf extract, which showed
excellent and encouraging results among all the extracts
studied in this research. Even low concentrations (5 ppm)
of crude extract were lethal on mosquito larvae.

International Journal of Research in Botany 2014; 4(1): 1-5

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 Aboli Lale and Dk Kulkarni (2010) reported the Therefore, among the 3 selected extract studied,
repulsion activity of Pongamia pinnata seed powder along the bark of Pongamia pinnata showed highest larvicidal
with the leaves, pod shell and cow dung with cow urine activity against Culex quinquefasciatus mosquito larvae
fermented mixture, which showed 90% repulsion of within a short time of 12 hrs.
mosquitoes. 6. Conclusions
There is no such research on the larvicidal activity The botanical insecticides are generally safer,
of the crude extracts of Pongamia pinnata bark. When this readily biodegradable non-toxic but active against the
extract was given even at low concentrations the mosquito insect pest and lack toxicity to higher animals and they do
larvae showed 100 % mortality rate. not leave any phytotoxic residues in the environment and
Table.3. The larvicidal activity of Pongamia active against insect pest.
pinnata leaf crude extract at different concentration is The biological activity of this plant extract may be
represented in Table: 5. the data shows that, 100 % due to various compounds, including tannins, phenolics,
mortality rate of larvae was observed at 25 and 50 ppm of terpenoids, flavonoids and alkaloids. These compounds
concentrations of crude extract after 48 hrs of the treatment. may jointly or independently contribute to produce toxic
And at the concentration of 100 and 200 ppm 100 % activity against the mosquito species.
mortality rate was recorded after 36 hrs. At low The present investigation suggests that these
concentrations of crude extract i.e. 10 and 5 ppm after 48 extracts can be considered as alternative insecticide
hours of treatment the % mortality rate of mosquito larvae products for mosquito control in breeding sites, especially
was decreasing to 80 and 50 % respectively, and no in cases where susceptibility is decreasing. These can be
mortality rate was observed for control. commercialized as botanical (Insect Growth Regulators)
IGRS such as methoprene.
Table 3: Mortality rates of Culex quinquefasciatus mosquito
larvae at different concentrations of crude extract of Pongamia The plant derivatives which are probable sources
pinnata leaves. of biologically active ingredients for mosquito control need
Conc. Time (hours) n=20 to be investigated for further studies.
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