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LARVICIDAL EFFECT OF AMPALAYA (Momordica charantia)
FRUIT JUICE ON Aedes MOSQUITO LARVAE
Amtul. Noor, Asunala. Rajesh Kumar, Chevagoni. Suresh, Doddaka. Ghnapti Krishna Sayogi,
Gollapally. Vikram Goud, Kosaraju. Narendra kumar, Koutharapu. Deepak Chandran, Pagadala.
Devi Vara Prasad, Rama. Annapurna, Rama. Mounika, Rama. Nikhil Raj, Sah. Sitesh Kumar,
Senguttuvan. Rahul Anand, Tamang. Nima, Uppugalla. Ruzu Rohith Reddy
Mrs. Mary Grace G. Villaflor
Abstract
This study was intended to determine if Ampalaya fruit juice can be used as larvicidal agent
for Aedes larvae and also compares its larvicidal property with that of a commercial larvicide
(ABATE 1SG). This research was experimental in nature, using completely randomized
design with commercial larvicide as positive control and pure water as negative control.
Aedes mosquito eggs were collected from one of the researchers residence, breeding and
hatching was done in the zoology laboratory of CDU. Two (2) to four (4) days old larvae were
used as our research subject. Beaker, Measuring Cylinder, Plastic Containers, Spoon were the
materials used in this research. After the setup of experiment, during each trial the numbers of
dead larvae were counted after time interval of 20 minutes, 40 minutes and one (1) hour. The
larvae that floated and did not respond to very slow stirring of the plastic spoon were
considered as dead larvae. Based on the result, it can be concluded that Ampalaya fruit juice
had a larvicidal effect, but it was inferior to the positive control ABATE 1SG. So, it might be
effective to other genera of mosquito larvae or other parts of Ampalaya may be more
effective.
Key words: Aedes larvae, Ampalaya fruit juice, Commercial larvicide
INTRODUCTION
Background of study
Mosquitoes are among the well-known
group of insect vectors that transmit
deleterious human diseases, which pose as
the major public health challenges eroding
development in the poorest countries of the
world (Awad and Shimaila, 9 (4) : 637).
-----------------------------------------------------
Sah. Sitesh Kumar
+069326640583
Their medical importance as vectors for the
transmission of serious diseases that cause
morbidity, mortality, economic loss and
social disruption such as malaria, filariasis,
yellow fever, dengue and other viral
diseases as well (Becker et al. 325).
The species of Aedes are important vectors
in the transmission of dengue, yellow fevers
and chikungunya fevers, and the like.
Dengue is a serious and ancient problem
spread by the bite of the infected Aedes
mosquitoes and its complications affects the
kidney, liver, brain and blood which could
be very fatal. Dengue fever is an actual
frequent epidemic viral disease transmitted
by the mosquito Aedes aegyptii. These are
the burning issue now-a-days. These health
conditions are endemic in the developing
countries of Africa, Latin America and Asia.
World Health Organization (88),
Brown (2 : 123- 140) and Lee et al. (44: 105
- 112), reported about the development of
physiological resistance to the chemicals by
mosquito species. Some mosquito species
developed resistance to malathion (Guneady
et al. 50 (1) : 45- 54), a conventional
pesticide, and to deltamethrin (Chen wen-
mei 33,(1) : 14 - 20) like the adult Culex
pipiens. These mosquitoes have developed
resistance against currently used
insecticides, thereby hindering their
effective control and possibility of
eradicating them in areas where they are
prevalent. Furthermore, the adverse effects
of conventional insecticides on the
environment and animals, including
humans, and the limited number of
available insecticides compel continued
search for safer plant insecticides and
larvicides that could be used in the control
of these vectors. Medicinal plant extracts
and their constituents have proved to be
biodegradable, have low mammalian
toxicity and induction of resistance while
their activities were similar to those of the
standard drugs, such as temephos and
methoprene.
These viral diseases and their
resistance to currently used insecticides
were the reasons that researchers decided to
study whether Ampalaya fruit extract could
also be a possible mosquito larvicide.
Theoretical framework
According to Bayer Environmental
Sciences and Reinert (16: 175- 188), there
are about 3500 species of mosquitoes,
grouped into 3 sub-families,
Toxorhnychitinae, Anophelinae, and
Culicinae. Although there are over 3500
species of mosquito through the world, all of
which live in specific habitats, exhibit
unique behavior and bite various animals.
Despite these different behaviors, all
mosquitoes share some common traits, such
as four stage life cycle. After a female
mosquito bites the animal and obtains the
blood, it lays eggs on the surface of stagnant
water, in a depression, or on the edge of a
container where rain water may collect and
flood the eggs. The eggs hatch in two to
three days and mosquito larvae or wriggler
emerges. Mosquitoes are most efficiently
and economically destroyed when they are
in the larval stage and are concentrated in
their breeding site (Ellis 9). The larvae of
Aedes mosquito were unable to survive in
water of a high degree of salinity due to
exosmosis. (Wigglesworth, V.B.).
The larvae live in the water, feed and
develop into the third stage of the life cycle
which takes four to ten days and is called a
pupa or tumbler. The pupa also lives in the
water but do not feed. Finally, the mosquito
emerges from the pupal case and it is ready
to bite as a fully developed mosquito
(Evelyn, 6).
Aedes bites only human beings.
Twenty-four to 48 hours following the
emergence of the adult mosquito from the
pupa and after mating has taken place, the
female seeks out the blood for egg
production in which it has two peaks for the
biting time, one at the dawn just after
sunrise and the second at the dusk before
the sunset. After female mosquito feeds on a
person whose blood contains virus, it can
transmit dengue either immediately or after
an incubation period of 8 10 days, during
which the virus multiplies (Evelyn, 7)
According to T. K. Lim (344-348),
Momordica charantia is tropical vine of the
family Cucurbitaceae widely grown for
edible fruit, which is among the most better
of all vegetables. An English name for the
fruit is bitter melon or bitter gourd. In the
Philippines it is commonly known as
Ampalaya, Hindi karela; Nepali- Tite
Karela; Tamil pakal, pavakka; Marathi
karke. The fruit has a distinct warty looking
exterior oblong shape. It is hollow in cross
section with relatively thin layer of flesh
surrounding a central seed cavity filled with
large flat seeds and pith. It grows in areas
where annual precipitation ranges from 480
mm to 4100 mm. All parts of the plant are
equally important as fruits of Ampalaya
(Momordica charantia) contain Charantin,
Polypeptide-p as its constituent elements
and also the leaves of the plant contains
Charantin, Polypeptide-p as the main
component whereas SEEDS contains
Charantin , Polypeptide-p along with Vicine
as its constituents.
Along with these components the
plant contains several biologically active
group, chiefly Momordican I, Momordican
II and Cucurbitacian B. The plants contains
also several bioactive glycosides (including
momordin, charatine, charnantitosides,
Goya glycosides, and momordicosides) and
other terpenoids compounds (including
momordicin 28, momordicinin,
momordicilin, momordenol and momordol).
It also contains cytotoxic (Ribosome-
inactivity) proteins such as momorcharin
and momordin. Also the aerial parts of the
Bitter gourd / Ampalya possess a number of
active ingredients including tannins,
flavonoids, alkaloids, quinines and phenol.
Fruit consist of amino acids such as Beta
alanine, Gamma Alanine, Glutamic acid,
Tryptamine and Gama amino-butyric acid
among others. Seeds also contain amino
acids and include Arginine, Aspartic acid,
Glycine, Leucine, Lysine and Histidine (T.
K. Lim,347-353). The juice extracted from
the fresh fruit of Momordica charantia has
been used in our research.
Review of related literature
Larvicides are chemical substances
or group of insecticides used to stop
mosquito larvae from maturing into biting
adults that transmit various diseases.
Dichloro-diphenyl-trichloroethane (DDT),
pyrethruim, heptachlor, diedrin and lindane
were the most commonly used larvicides in
the past to achieve these control measures.
The commonly and repeatedly used
larvicides are fuel oils, kerosene and
insecticide formulations (Truman et al.,
207). Synthetic organic larvicides, although
very efficacious to target species such as
mosquitoes could be detrimental to a variety
of animal life including man.
NICC reported that
organophosphate temphos which is a
Larvicide usually used in breeding site,
though slightly toxic to target organism,
may cause headache, loss of memory and
irritability to man. Besides, the incessant
use of chemical insecticides had often led to
the disruption of natural biological control
system, and outbreak of insect species as
noted by (Chaithong et al., 31(1): 138- 144).
Moreover, these chemicals could be
carcinogenic, mutagenic and teratogenic.
Many plant juices could be used to
drive mosquitos away, eg. Eucalyptu
citriodora, Pterigeron odorus, Santalum
lanceolatum, Momordica charantia.
(Zohara and Uriel, 183). Larvae can be
killed by keeping it into hypertonic solution
as due to the difference in pressure gradient
and concentration gradient osmosis can take
place and then cell loose its water shrink
and dies. ( Biology, p.180). Plants that were
termite or insect resistant, were used as fish
poison and folkloric ally in treating malaria
and fever as well as those with reported
insecticidal, mosquito repellent and
mosquitocidal activities had demonstrated
promising larvicidal activities . However,
no co-relation had been established between
larvicidal activities and these properties but
an earlier study with a common medicinal
and vegetable plant of Momordica
charantia Linn (Family: Cucurbitaceae),
had shown the insecticidal activity of this
plant against mustard saw fly. (T.K. Lim,
356)
The larvicidal nature of fleshy fruit
wall of Momordica charantica Linn
(Family: Cucurbitaceae) was found first
time, in the management of mosquitos done
in the year 2009 November by Batabya et
al. ( P.1205-10) in view of the recently
increased interest in developing plant based
insecticides as an alternative to chemical
insecticides. This study was undertaken to
know the larvicidal potential of the various
fruit juices of Momordica charantia
(Cucurbitaceae) against culex species of
mosquito vector.
Significance of study
This research could help the Rural
and Community Health Workers to enhance
rural based projects promoting locally
grown plant for medical purpose which
could serve as the cheaper alternative to
expensive drugs available in the market.
This research would also help in finding the
active ingredient in Ampalaya fruit and
potentially spearhead the manufacturing of
commercial larvicides that would benefit
the people. The farmers could gain profit by
cultivating this plant as cash crops.
Industries could probably get benefitted
from this study since they could produce
new larvicides from local resources. The
botanists could also be benefitted as they
would get new information about this plant.
This would create an interest towards
further research and exploration.
Government would also be equally
benefitted by exporting larvicides and
nature would also be free and fresh since
this juice is natural and biodegradable
unlike some other commercial larvicides
which are non-biodegradable.
Objectives
General objectives
The study aimed to determine the
larvicidal effect of Ampalaya (Momordica
charantia) fruit juice on Aedes mosquito
larvae and also compare its larvicidal nature
with commercial larvicide (ABATE 1SG).
Specific objective
This study aimed to:
1. keep a tally of number of Aedes mosquito
larvae killed after 20, 40 and 60 minutes of
exposure to
A) Ampalaya fruit juice in tap water
a.1) 25%
a.2) 50%
a.3) 75%
B) commercial larvicide (ABATE
1SG)
2. Compare the number of Aedes mosquito
larvae killed by the administration of
Ampalaya fruit juice and commercial
larvicide (ABATE 1SG).
3. Determine the significant difference in the
proportion of dead wrigglers between
Ampalaya fruit juice and commercial
larvicide (ABATE 1SG).
Scope and limitation
This experimental study involved
the determination of the larvicidal effect of
various concentrations of juice extracted
from the fruit of Ampalaya on the two (2)
four (4) days old larvae of Aedes. The
determinations of chemical composition of
the juice were not included in the study. The
concentrations of Ampalaya fruit juice
below 25% and above 75% were not
considered in the experiment. The collected
eggs were of Aedes mosquito as the eggs
were laid separately, were elongated and
black in color. But, the species of the Aedes
mosquito were unidentified.
Hypothesis Statement of Null Hypothesis (Ho) There was no significant difference
in the proportion of dead wrigglers between
Ampalaya fruit juice as Aedes mosquito
larvicide and a commercial larvicide.
Statement of Alternative Hypothesis (Ha)
There was a significant difference in
the proportion of dead wrigglers between
Ampalaya fruit juice as a mosquito larvicide
and a commercial larvicide.
MATERIALS AND METHODS
Research design: The study was
experimental in nature, using Single
Variable completely randomized design to
determine the larvicidal effect of
Momordica charantia fruit juice (variable)
on Aedes mosquito larvae (constant), which
were selected and assigned randomly. Also
it was compared with commercial larvicide
(ABATE 1SG) as positive control and water
as negative control.
Research locale: The preparation of the
different concentrations of Momordica
charantia fruit juice was conducted in Cebu
Doctors University research laboratory.
The collection of mosquito eggs was
conducted at one of the researchers
residence located near CDU, Mandaue City.
The breeding and hatching of the eggs were
conducted at Cebu Doctors University,
zoology laboratory. This area was suitable
for conducting experiment as it is well
lighted, well-furnished and well equipped
with the materials needed for experiment.
Research subject: The research subjects
were two (2) to four (4) days old Aedes
larvae i.e. 2nd -3rd instar larvae which were
randomly selected from the basin with
naturally bred and hatched larvae.
Collection of sample
Mosquito larvae collection
White plastic containers coated in
black were used as breeding site of Aedes
mosquito. The containers were lined with
filter papers and half filled with clean water.
The plastic containers with filter paper were
placed in the area where Aedes mosquitoes
naturally bred such as human surrounding.
The containers were checked every morning
for the presence of mosquito eggs. When
the tiny black patches were seen on the
filter papers, the filter papers were removed
from the black container, air dried and were
stored into a clean and dry container. The
larvae were then brought to Dr. Emmanuel
Pardian for authentication of
identification.
Hatching of Aedes Eggs
The transparent plastic containers
were filled with clean water. The filter
paper with eggs were cut into pieces and
then placed in the plastic containers to
allow the hatching of eggs. The set ups were
checked daily for the hatching of the eggs
and once the larvae were seen, they were
transferred into another plastic container
and labeled accordingly as to how many
days old. After the collection of desired
amount of larvae (between one (1) to four
(4) days), they were transferred to nine (9)
different cups for experimental setups (three
(3) for each concentration), three (3) for
negative control and three (3) for positive
control. Using a plastic spoon, six larvae
were randomly selected and assigned in
each of the fifteen (15) cups.
Procedure of extraction
The selected, fresh and clean fruits
of Momordica charantia were bought from
the mall and brought to Mr. Vitaliano
Fernandez for authentication of
identification. After confirming that they
were really Momordica charantia, they
were then brought into the research
laboratory for the extraction process. Nearly
three and half (3.5) kilograms of fresh fruits
of Momordica charantia were washed and
chopped into small pieces. Then, they were
crushed in a mixer. The juice was then taken
out and filtered using a clean cloth. Then
three (3) different concentrations of juice
were prepared by mixing it with proper
amount of water. For the 25%
concentration, 25 ml of pure juice and 75
ml of water were mixed, for 50%
concentration, 50 ml pure juice and 50 ml
were mixed and for the 75% concentration,
75 ml pure juice and 25 ml water were
mixed. The prepared different
concentrations of juice were used in the
experiment along with water as negative
control and larvicide (ABATE 1SG) as
positive control.
Data collection: The experiment was
conducted on the 6th of December 2013 in
the zoology laboratory in CDU. The three
plastic cups were taken and filled with 25
ml, 50 ml and 75 ml of pure water. The
three different concentrations were obtained
by adding 75 ml, 50 ml and 25 ml of pure
Momordica charantia fruit juice
respectively. As for the negative control,
100 ml of pure water was kept in one cup.
Then six (6) naturally bred larvae were
placed in each cup and the cups were kept
on the table. The set ups were checked
every 20 minutes until one (1) hour. A stop
watch was used to note down the duration
of time. Same steps were followed for three
(3) trials along with positive and negative
control set ups.
At the end of each trial, the numbers
of dead and alive larvae were noted. The
larvae that floated and did not show any
movement or even did not respond to very
slow stirring of the plastic spoon were
considered dead. The number of dead and
alive larvae were counted and recorded in
the tabulated data sheet.
Definition of terms
Aedes Mosquito- Aedes mosquitoes are
typically small mosquitoes. They usually
have black and white stripes marking on the
body and legs. They usually bite only
during day time.
Larvae - larvae are the distinct juvenile
forms of many insects which undergo
complete metamorphosis. It is the feeding
stage of the insect development. It is
constant.
Larvicide - An agent for killing larvae, in
this experiment, it is the Ampalaya juice.
Commercial larvicide - Insecticide that is
specially targeted against larval stage of any
insects, in this experiment, ABATE 1SG.
Ampalaya fruit juice - It is the juice
extracted from the fresh Ampalaya fruit
with the help of grinder. After the extracted
of juice, desired amount of water is added
to make different concentration (25%, 50%
and 75%). It is variable.
RESULTS AND DISCUSSIONS
Result
The tables below showed the summary
of the data gathered from the research and
the Mean of the different concentrations.
Table 1 Percentage (%) of dead Wrigglers
A) After 20 minutes
Based on the result, it can be seen that the
ABATE 1SG is 50% effective after 20
minutes whereas highest concentration of
Ampalaya fruit juice is only 11.11%.
B) After 20 minutes
Based on the result, ABATE 1SG was
66.67% effective after 40 minutes whereas
25%, 50% and 75% concentrations of
Ampalaya fruit juice were comparatively
less effective and showed 5.56%, 22.22%
and 27.78% effectiveness respectively.
Substance used
No. of Trials
ABATE 1SG
25% of Ampalaya juice
50% of Ampalaya juice
75% of Ampalaya juice
Trial 1 50.00 0.00 0.00 16.67
Trial 2 66.67 0.00 16.67 16.67
Trial 3 33.33 0.00 0.00 0.00
MEAN 50.00 0.00 5.56 11.11
Substance used
No. of Trials
ABATE 1SG
25% of Ampalaya juice
50% of Ampalaya juice
75% of Ampalaya juice
Trial 1 66.67 16.67 33.33 33.33
Trial 2 66.67 0.00 16.67 33.33
Trial 3 66.67 0.00 16.67 16.67
MEAN 66.67 5.56 22.22 27.78
C) After 1 hour
Substance used
No. of Trials
ABATE
1SG
25% of
Ampalaya
juice
50% of
Ampalaya
juice
75% of
Ampalaya
juice
Trial 1 83.33 33.33 50.00 66.67
Trial 2 83.33 16.67 33.33 50.00
Trial 3 100.00 16.67 33.33 50.00
MEAN 88.89 22.22 38.89 55.56
As shown in the above table, the ABATE
1SG was more effective after 1 hour as
compared to 25%, 50% and 75%
concentrations of Ampalaya fruit juice.
Table 2
Comparison of Results
A) After 20 min
Group No. of Trials
Average % of dead Wrigglers
P value
Remarks
With ABATE 1SG 3 50.00 0.038 Since the P < 0.05 level of significance, the null hypothesis (Ho) was rejected.
With 25% of Ampalaya fruit juice
3 0.00
With 50% of Ampalaya fruit juice
3 5.56
With 75% of Ampalaya fruit juice
3 11.11
With ABATE 1SG 3 50.00 0.000 The Ho was rejected because the P value is < 0.05 level of significance.
With 75% of Ampalaya fruit juice
3 11.11
Based on the given results, the P value
(0.038) was less than 0.05 level of
significance. Hence, the null hypothesis
(Ho) was rejected. It implies that the number
of dead wrigglers using Ampalaya fruit juice
in different concentrations and using
ABATE 1SG were significantly different.
More dead wriggler were found when
ABATE 1SG was used. Hence, Ampalaya
fruit juice is not an effective mosquito
larvicide.
B) After 40 min
Group No. of Trials Average % of dead Wrigglers
P value
Remarks
With ABATE 1SG 3 66.67 0.027 Since the P < 0.05 level of significance, the null hypothesis (Ho) was rejected.
With 25% of Ampalaya fruit juice
3 5.56
With 50% of Ampalaya fruit juice
3 22.22
With 75% of Ampalaya fruit juice
3 27.78
With ABATE 1SG 3 66.67 0.000 The Ho was rejected because the P value is < 0.05 level of significance.
With 75% of Ampalaya fruit juice
3 27.78
In this table it can be seen that the
percentage (%) of dead larvae was only
27.78% for the 75% conc. of Ampalaya fruit
juice. The P value at 40 minute is 0.027,
which is less than 0.05 level of significance.
Hence, the null hypothesis (Ho) was
rejected. The results also show that the
number of dead wrigglers using Ampalaya
fruit juice in different concentrations and
ABATE 1SG differed significantly even
after 40 minutes.
C) After one (1) hour
Group No. of Trials Average % of dead Wrigglers
P value
Remarks
With ABATE 1SG 3 88.89 0.020 Since the P < 0.05 level of significance, the null hypothesis (Ho) was rejected.
With 25% of Ampalaya fruit juice
3 22.22
With 50% of Ampalaya fruit juice
3 38.89
With 75% of Ampalaya fruit juice
3 55.56
With ABATE 1SG 3 88.89 0.000 The Ho was rejected because the P value is < 0.05 level of significance.
With 75% of Ampalaya fruit juice
3 55.56
Based on the above results, the percentage
(%) of dead larvae was only 55.56% for the
75% conc. of Ampalaya fruit juice. Here, the
P value after one (1) hour was 0.020 which
is less than 0.05 level of significance.
Therefore, the null hypothesis (Ho) was
rejected. More dead wrigglers were found
by treating with ABATE 1SG (88.89).
Hence, the number of dead wrigglers using
Ampalaya fruit juice in different
concentrations (even at 75% concentration)
and ABATE 1SG were significantly
different after one (1) hour.
Discussion
Based on the experiment, it was seen
that more number of larvae died in the
highest concentration of Ampalaya fruit
juice probably because of hyper tonicity
(Biology, p.180) of the solution as the
tonicity of Ampalaya fruit juice was more
compared to cells of the larvae, exosmosis
took place eventually resulting in their
death.
An experiment performed on Aedes
mosquito larvae showed that the larvae of
Aedes mosquito were unable to survive in
water of a high degree of salinity due to
exosmosis. (Wigglesworth, V.B. )
However, few larvae died in the
lowest concentration of Ampalaya fruit
juice. This showed the presence of certain
chemicals in the Ampalaya fruit juice that
had some larvicidal property. Therefore
Ampalaya fruit juice had larvicidal effect on
Aedes larvae even though it is minimal.
Some researchers have shown that many
plant juice like Eucalyptus citriodora,
Pterigeron odorus, Santalum lanceolatum,
Momordica charantia were used to drive
mosquitoes away (Zohara and Uriel, 183)
but Ampalaya fruit juice did not prove to be
as effective as them.
Some researches (Maurya Para et
al., 2009) have also shown the larvicidal
nature of fleshy fruit wall of Momordica
charantia to Culex mosquito larvae.
Thus, it can be said that the
larvicidal property of Ampalaya fruit juice
is selective to Culex mosquito larvae as
proven by the work of (Batabyal L et al.
para.1) while not so effective in the case of
Aedes mosquito larvae.
CONCLUSION
In conclusion, the Ampalaya fruit
juice had a larvicidal effect, but it was
inferior to the positive control ABATE 1 SG.
RECOMMENDATION
From this very research, it was
found out that the larvicidal property of
Momordica charantia fruit juice was
minimal. Even the 75% concentration
solution was not very effective in killing
Aedes mosquito larvae. However, the other
parts of Momordica charantia plant such as
leaves, root, stem etc. might be useful in
killing the Aedes mosquito larvae. Further
research using other parts of Ampalaya was
therefore recommended for further study if
they have larvicidal effect.
As mentioned before, the Ampalaya
fruit juice has minimum larvicidal effect
when it comes to Aedes mosquito larvae.
But, it might be effective in killing
mosquito larvae of other genera such as
Anopheles, Culex. It can also be tried for
different species of mosquitoes.
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1976.
Zohara Xaniv & Uriel Bachrach. Handbook
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