Journal of Mosquito Research 2015, Vol.5, No.12, 1-7
5
grinder. For solvent extraction procedure 200 gm of
finely ground powdered fruit were extracted with
ethyl acetate for 72 hours, in a grease free Soxhlet
apparatus. After collection and filtration, the extract
was subjected to evaporation in a vacuum rotary
evaporator below 40
°
C, and then the extract was
stored in a refrigerator within air tight glass container
until further use.
4.4 Test mosquitoes
Adult
Cx. quinquefasciatus
and
An. stephensi
mosquitoes
were collected from the mosquito colonies maintained
in pathogen free and hygienic condition in the laboratory
of Mosquito, Microbiology and Nanotechnology Research
Units, Department of Zoology, The University of
Burdwan. Adult mosquitoes were reared in humidified
cages and fed with 10% aqueous glucose solution.
Female mosquitoes were periodically blood-fed on
restrained albino rat for egg production.
4.5 Adulticidal bioassay with the fruit extracts
This bioassay was performed according to
protocol with minor modifications. Appropriate amount
of residue of ethyl acetate extract was dissolved in
2.5 ml acetone to achieve solutions of desired
concentrations (50, 100, 150, 200, 250 ppm) and
applied on Whatman no. 1 filter papers (size 12 ×15
cm
2
) (Dua et al., 2008). For experimentation with
crude extract 2.5 ml juice extracted from fresh mature
fruits was applied on the filter papers. Control papers
were treated only with acetone. After drying the filter
papers were kept inside plastic holding tube covering
the inner side of the tube. Separate batches of 25
An.
stephensi
and 25
Cx. quinquefasciatus
adult mosquitoes
(2–5 days old blood starved, glucose fed) were cautiously
transferred into a plastic holding tube and they were
allowed there for acclimatization till 1 hour. Then the
mosquitoes were exposed to the treated papers containing
holding tube for 1 hour. They were transferred back to
the previous holding tube and kept there for 24 hour
for recovery. Mosquitoes were provided with 10%
glucose solution soaked in cotton ball. Control
experiments were performed similarly. The test was
repeated four times for each mosquito species. Adulticidal
efficacy of the plant extracts were expressed in terms
of percent mortality of the mosquitoes after 24 hour of
recovery period.
Percent mortality was corrected by using Abbott’s
formula (1925).
4.6 Repellency potentiality test with the fruit
extracts
Repellent activity test of plant extract was executed
through percentage of protection in relation to dose
method. One hundred (3-4 days old) blood starved
female mosquitoes were kept inside two net cages (45
x 30 x 45 cm
3
) for concomitant test and control
experiments. A stock solution of 50 ml of 1000ppm
concentration was made by dissolving the ethyl
acetate extract in ethanol. A number of ranges of test
solutions (1%, 25%, 50%, 75% and 99%) of the ethyl
acetate extract were prepared by dissolving the stock
solution in ethanol. The forehands of the volunteer
(self volunteer) were cleaned with ethanol. Different
test solutions were applied thoroughly from the elbow
to the tip of the fingers on one hand followed by
drying (test for each concentration was done separately).
The other forehand was treated with ethanol only to
serve as control. After gently tapping the sides of the
bioassay cage to stimulate the mosquitoes, the treated
and control forehands were put into the cages first and
kept there for 5 min. If at least two mosquitoes landed
on the test areas, the hands were shaken off before
imbibing any blood and withdrawn from the cages.
The number of bites was counted over 5 min/hour
from 6.00 p.m to 6.00 a.m. Care was taken between
the testing periods to minimize contact of the test sites
with any other influence. All the experiments were
conducted four times with control. Repellency of the
crude extract was also assessed by applying it directly
on the forearm without preparing any graded
concentration. In that case the other forearm was
washed with distilled water to serve as control. The
percentage protection was calculated by using the
following formula (Venkatachalam and Jebanesan, 2001)
Conflict of Interest
We declare that we have no conflict of interest.
Acknowledgements
The authors gratefully acknowledge the financial support
provided by UGC [No.F.17-8/08 (SA-I)] and UGC-DRS. They
are indebted to Professor Dr. A. Mukhopadhyay, Botany
