LARVICIDAL EFFECT OF AMPALAYA (Momordica charantia) FRUIT JUICE ON Aedes MOSQUITO LARVAE

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

[email protected]

+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



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




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


3 5.56


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.


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



3 5.56


3 22.22


3 27.78



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



3 22.22


3 38.89


3 55.56



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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Medicinal Plants. Springer Dordrecht

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Truman, et al. Scientific Guide to Pest

Control Operations 3rd ed. Cleveland

Ohio:Harvest Publishing Company,

1976.

Zohara Xaniv & Uriel Bachrach. Handbook

of Medicinal Plants. New York: Food

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LARVICIDAL EFFECT OF AMPALAYA (Momordica charantia) FRUIT JUICE ON Aedes MOSQUITO LARVAE