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JOURNAL OF MOSQUITO RESEARCH
95
concentration) for the bioassay experiment.
3.4 Differential solvent extraction
For solvent extraction, fresh and clean leaves of
A.
aspera
were dried for few days in shed. 200 g dried
leaves of
A. aspera
were put into the column of the
Soxhlet apparatus while 2 lit solvent was laden into
the solvent chamber following 1:10 ratio. Six different
solvents in a non-polar to polar fashion viz. petroleum
ether, n-hexane, ethyl acetate, chloroform: methanol
(1:1 v/v), acetone, and absolute alcohol were passed
through the column one after another. The extraction
period was fixed for 72 hours with 8 hours maximum
a day for each solvent. Elutes were collected from the
solvent chamber and concentrated through
evaporation in a rotary evaporator. The extractives
were preserved at 4
℃
in a refrigerator.
3.5 Larvicidal bioassay
Trusting the WHO standard protocol (WHO/ VBC,
1981) the larvicidal bioassay was executed at the
Mosquito and Microbiology Research Units,
Parasitology Laboratory, The University of Burdwan.
Twenty five larvae of precise stage from the
laboratory reared larval colony were re-located in
Petri-dishes of 9 cm diameter (150 mL capacity) and
filled with 100 mL of tap water. Crude extractives
were added in a graded concentrations ranging from
0.1% to 0.5% in different Petri dishes while 20 ppm to
100 ppm solvent extracts were taken in different Petri
dishes for executing larvicidal bioassay. Each
experiment was completed in triplicate (n=75) with a
set of controls. Petri dishes were kept at room
temperature (28 ± 2
)
℃
, within (88±2)% relative
humidity range for a total observation period of 72
hours. The larvae were assumed dead when they
didn’t exhibit any movement by the pricking with a
sharp needle in the siphon or cervical region or they
were unable to achieve the water surface
(
Macedo et
al., 1997)
.
The no. of deceased wrigglers was
calculated every 24 hours period up to 72 hours and
percentage mortality was recorded from the average
value of three replicates.
The mortality data of 48 h
and 72 h were expressed by additions of the
mortalities of 24 h and 48 h, correspondingly. Firstly
all the solvent extractives were screened against 3
rd
instars larvae and then the experiment was elaborated
with the best active fraction against all the instars.
3.6 Costing the impacts on non-target populations
The little creatures sharing the same environment with
mosquitoes are considered as the most fatal risk group.
Vulnerability of these organisms to leaf extractives
was figured out on
Chironomus circumdatus
larvae
(insect). They were exposed to concentration level of
LC
50
value of 24 h of 3
rd
instars larvae to examine the
mortality and other irregularities such as tardiness of
swimming activity up to 72 h of exposure.
3.7 Statistical analysis
The percentage mortalities (%M) were precised by
Abbott’s formula (Abott WS, 1925) during the
observation of larvicidal potentiality of the plant
extracts. Judicious determination of LC
50
and LC
90
values of crude and solvent extracts were carried out
through Log-probit and regression analyses. Further
statistical justifications were elaborated through
ANOVA analyses using different instars, hours and
different concentrations as three random variables to
validate the significance between the above
parameters and larval mortality.
Acknowledgements
The authors are indebted to Professor Dr. A. Mukhopadhyay,
Botany Department, The University of Burdwan, for his kind
help in plant species identifications. We are grateful to UGC
DRS and DST-INSPIRE for providing financial assistance.
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Journal of Mosquito Research