Lemongrass (Cymbopogon citratus), Garlic (Allium sativum) and Papaya (Carica papaya L.

Leaf: Botanical Mosquito Culicidae Larvicide

A Research Plan

(LIFE SCIENCE- TEAM CATEGORY)

Gracelle G. Lucero

Llainz Leirrence G. Abapo

Althea Thyrese M. Zurita

Luciano B. Rama Sr. Memorial National High

School Esperanza, Poro, Cebu

Cristina P. Guntiñas

Adviser

1
 Table of Contents

Title Page 1

Table of Contents 2

List Figures and Tables 3

Rationale 4

Research Question or Problem 8

Goals/ Expected Outcome/ Hypotheses----------------------------------------------9

Materials Used/ Preparation 10

Procedures 11

Risk and Safety 16

Data Analysis 17

Bibliography 21

2
 List Figures and Tables

Table 1: Materials to be Used in the Experimentation......................................................10

Table 2: Variables of the Study.........................................................................................11

Figure 1: Flow of the Study...............................................................................................12

Table 3: Distribution of Larvae Samples and....................................................................17

Assignment of Concentrations

Table 4: Observation of Mosquito Larvae after...............................................................18

the Application of Natural Larvicide

Table 5: Mortality Rate of Mosquito Larvae.....................................................................19

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 Rationale

Mosquitoes are thin, long-legged, two-winged insects and are typically six to 12

millimeters in length. Both males and females have antennae and an elongated "beak" or

proboscis three to four times longer than its head. These insects belong to the Diptera order,

known as the true flies, in the family Culicidae. All true flies have two wings; however,

mosquitoes are the only true flies to have scaled wings. Their way of breeding is by laying their

eggs on the surface of fresh or stagnant water. Eggs can be laid anywhere that water remains

stagnant — in cans, discarded tires, bird baths, ditches or puddles, although mosquitoes prefer

water surfaces protected from wind. It doesn’t take a lot of water as some mosquitoes can breed

in as little as one centimeter of standing water. Culex mosquitoes usually lay their eggs at night,

and they stick together due to a special sculpture on the sides of the eggs that form a raft

approximately 64 millimeters long and 38 millimeters wide. The raft can contain anywhere from

100 to 300 eggs and looks like a flake of soot floating on the water surface. Anopheles species

lay their eggs singly on the water surface. Aedes mosquitoes also lay their eggs singly, and most

species select damp soil. Aedes eggs are more resistant to drying out than those of other genera

and some require low temperatures before the eggs will hatch. Others hatch only when flooded

with water above a critical temperature. Certain mosquitoes can carry and spread disease-causing

bacteria. These organisms (disease pathogens) are frequently viruses that are caught or "picked

up" by the mosquito when it bites (feeds on) an affected host. Domestic and wild animals, as

well as humans, serve as hosts. The virus grows and replicates within the mosquito. When an

infected mosquito bites another, uninfected host, the virus is transmitted through its saliva. The

mosquito works as a "vector" of the sickness by transmitting the virus (Stephen Higgs, 2005).

4
 For hundreds of years, mosquitoes have been recognized as the essential arthropod

vectors for the spread of pathogens that kill millions of people worldwide each year. Some of the

infectious diseases spread by mosquito bites includes the West Nile virus, yellow fever, dengue

fever, filarial, and malaria, which can eventually prove fatal to people (Sharma, 2001). Malaria

was estimated to have caused 216 million infections and 4, 45,000 deaths worldwide in 2016.

Furthermore, 100 million people in Africa and Southeast Asia suffer dengue fever each year and

predicts that 1 billion people worldwide will be at risk of catching the disease in the next 50

years. Bangladesh is especially vulnerable to dengue outbreaks due to high temperatures and

humidity, which are mostly caused by rising urbanization and inconsistent seasonal rain. Acute

dengue symptoms include fever, muscle pains, headaches, and a rash (WHO, 2020).

The World Health Organization estimates that more than 2.5 billion people live in

dengue- endemic areas, which contribute significantly to an estimated yearly death toll of 25,

000.

In the Philippines the most common disease caused by mosquitoes is Dengue. This

disease is epidemic in the Philippines and considered one of its eight pervasive infectious

diseases. From 2008 to 2012, the Philippines' Department of Health (DOH) reported 585,324

dengue cases, with a case fatality rate (CFR) of 0.55% or 3,195 deaths. Among the 10

Association of Southeast Asian Nations (ASEAN), the Philippines ranks fourth in the number of

dengue cases. Four dengue virus (DENV; Flavivirus) serotypes are circulating in the

Philippines8, transmitted by Aedes aegypti L. and Ae. albopictus Skuse (Diptera: Culicidae).

Environmental risk factors and inconsistent preventive practices, in addition to urbanization,

increasing population, inadequate public health infrastructure, poor solid waste management, and

lack of an effective mosquito surveillance system contribute to the growing dengue challenge.

5
 Different localities in the country recognize the threats brought about by these diseases

and consider different strategies in looking after the welfare of the general population. The risk

of being bitten by mosquitoes among children is very high, especially that mosquitoes can thrive

abundantly in environments if artificial containers can act as their breeding habitats. Children in

schools are recognized as important vulnerable groups in the Philippines, as their burden of

mosquito-borne diseases is great as they may be hospitalized, suffer long-term disabilities, incur

foregone opportunities by missing out in school, and, in worse conditions, even die. There is a

need to monitor the occurrence of mosquito vectors in places where children meet and

congregate and to institute vector surveillance strategies so that appropriate preventive and

control measures may be put in place to protect the children’s public health and welfare (Mistica,

Ocampo, De Las Llagas, Bertuso, Alzona & Magsino, 2019).

When compared to the same period last year, the Department of Health (DOH) in region

7 observed a 118 percent rise in dengue cases this year. The Department of Health previously

proclaimed a nationwide dengue epidemic in response to the 146,062 dengue cases documented

between January and July 20 of this year, which is 98 percent more than the cases recorded

during the same time last year (Baladya, 2019).

Such disease outbreaks impose a social and economic cost while obstructing economic

growth through several channels, including quality of life, absenteeism, incapacity to work, loss

of labor productivity, premature mortality, loss of savings, and medical costs, all of which result

in additional economic burden, that is, costs directly or indirectly related to disease diagnosis,

treatment, and prevention (Castro et al., 2017).

Managing the rising number of illnesses caused by mosquitoes entails controlling the

mosquito population. Larvicide products can be used to control the number of mosquitoes. For

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decades, interest in developing natural, environmentally friendly larvicides has significantly

increased. Synthetic larvicide poses many risks to humans, the environment, and other livestock.

The researchers ought to find alternatives for this synthetic larvicide.

In this study, the researchers want to develop an effective organic larvicide out of

Lemongrass (Cymbopogon citratus), Garlic (Allium Sativum), and Papaya (Carica papaya):

Lemongrass (C. citratus) has known for its toxic effect on mosquito larvae. This effect relates to

the citronella chemical compound inside the stalk and leaves, which reaches 35% and becomes

the densest chemical compound inside lemongrass. Citronella affects the mosquito through direct

contact. The mosquito in contact with citronella would lose its body fluid and die. C. citratus has

no effect on first instar larvae but did inhibit growth and increase mortality in A. aegypti’s later

developmental stages. In this study, lemongrass leaves were extracted using methanol. The

extracts were tested against fourth instar A. aegypti larvae to observe its larvicide effect.

(Mariam, Oktiviyari, Harahap, 2021).

Garlic is thought to be played by Allicin, and Diallyl disulfide has bactericidal and

bacteriostatic properties. Allicin works by interfering with the synthesis of parasite cell

membranes so the parasites cannot grow further. Allicin is also toxic to the parasite and bacterial

cells. The oil content in the garlic solution may change the surface tension of the water so that

the larvae have difficulty taking oxygen from the water surface. This is thought to cause the

larvae not to get enough oxygen to grow, leading to larval death. (Rahmah, Ashari, Suryadi,

Chairani, 2019)

Based on a study conducted (Malathi & Vasugi, 2015), Carica papaya is claimed to have

mosquito larvicidal and repellant properties against the mosquito Aedes aegypti. The

composition of Carica papaya that is responsible for repelling insects, particularly mosquitoes,

is the alkaloid
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content, which contains carpaine. This substance harms and attacks the neurological system of

insects. (Ma’ruf, Shoolihah, Darmono, Setiyawa, Yanuar Fadlullah, 2023)

The utilization of Lemongrass, Garlic, and Papaya as homemade larvicides offers several

advantages. Compared to synthetic chemicals, they have lower costs, are simpler to use, and

offer less environmental dangers. Additionally, by using these common substances, communities

that have little access to commercial larvicides can apply easy and long-lasting techniques to

lower mosquito populations and the dangers of disease transmission. By investigating the

larvicidal efficacy of lemongrass, garlic, and papaya leaf, this research addresses the need for

alternative and environmentally friendly approaches to mosquito control.

The findings of this study will assist in the development of homemade larvicide that can

be easily adopted by communities for effective mosquito population management. The study's

ultimate purpose is to advance sustainable and practical techniques for preventing mosquito-

borne diseases by providing informative information on the potential use of ordinary household

items as homemade larvicides.

This output to be generated in this study would be extremely beneficial in reducing the

rising population of mosquitoes in Barangay Esperanza, Poro, Cebu. This study is also

advantageous to local farmers because lemongrass, garlic and papaya may be cultivated in our

domestic fields, in which the government and the non-government agencies might provide with

financial assistance.

Research Question or Problem

Due to the continues neglect of people in managing areas where mosquito larvae are

rampant, the growing number of mosquitoes caused a lot of diseases, including dengue. This

study seeks to examine the effectivity of a product made through the extracts of lemongrass,

papaya
8
leaves and garlic which could serve as a useful tool to diminish the larvae which in extent lessens

the growing number of mosquitoes.

Research Questions:

1. How long will each concentration (2.5%, 5%, 10%, 25%, 50% and 100%) make lethal

effect on the collected larvae in each experimental setup?

2. What is the mortality rate of larvae of each experimental setup after the application of

the six larvicide solutions with the concentration of 2.5%, 5%, 10%, 25%, 50% and

100%?

3. Which among the six concentrations of natural mosquito (Culicidae) larvicide solutions

made from botanical extracts such as lemongrass (Cymbopogon citratus), garlic (Allium

sativum) and papaya (Carica papaya) can kill the greatest number of mosquito larvae in

the shortest period of time?

Goals/ Expected Outcome/ Hypotheses

This study’s ultimate goal is to improve sustainable and practical strategies for mosquito-

borne disease prevention by giving instructive information on the possible use of common

household products as homemade larvicides.

Null hypothesis: The concentrations of the extracts administered to the larvae had no

effect on the number of larvae after 24 hours.

Alternative Hypothesis: The concentration of extract applied has the potential power of

killing mosquito larvae.

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 Materials Used/ Preparation

As this study involves experimentation on the application of the six concentrations of the

prepared natural larvicide solutions to come up with a concentration that would best kill a

mosquito larva, this study requires different laboratory tools and other materials and resources to

carry out the experimentation procedure. (Please see Table 1 below.)

MATERIALS USAGE

NEEDED

Dipper Used in collecting larvae samples desired in the study.

Ladle Used in putting the larvae samples in the container from the dipper.

Container Used in storing the larvae samples after being collected.

Petri dish Used in storing the larvae samples during the experiment.

Knife Used in chopping the ingredients into smaller pieces.

Cutting board Used in placing the ingredients while being chopped.

Mortar & Pestle Used in crushing the ingredients into the ideal consistency of the extracts

after being chopped.

Gloves Used in protecting your hands from directly touching the ingredients.

Thin cloth Used in squeezing out the extracts from crushed ingredients.

Beaker Used in storing the extracts after being extracted.

Tablespoon Used in adding water to the extracts in the right amount.

Graduated cylinder Used in measuring the solute and solvent in coming up with the right

solution for the study.

Funnel Used in transferring liquids and not waste any solutions.

Table 1: Materials To be Used in the Experimentation

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 The table below presents the variables of the study which include the independent

variable that is the application of the different concentrations of the natural larvicide solution

made from the extracts of lemongrass, garlic and fruit-bearing papaya leaf. While the lethal

effects or the number of larvae died in each experimental setup (A, B, C, D, E, F) after the

application of the different concentrations (2.5%, 5%, 10%, 25%,50% and 100%) of larvicide

solution is considered the dependent variable of the study.

Variables Existing in the Usage

Study

Lemongrass (Cymbopogon citratus) The ingredients to be use in the formulation of six

Garlic (Allium Sativum) different concentrations (2.5%, 5%, 10%, 25%, 50% and

Papaya Leaves (Carica papaya) 100%) of the natural larvicide in caryring out of the

study.

Experimental Unit Usage

Mosquitoes Culcidae Larvae The samples to be experimented using the six

formulated concentrations of the natural larvicide in the

conduct of the study.

Table 2: Variables of the Study

Procedures

The following are the procedures to be undertaken in conducting the study. It involves

three stages of the development and operation that the researchers must achieve. This specifically

entails the flow of the study and the things that must be carried out. The table presents the stages

to be implemented.

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 Stage 1

1. Making of different solutions of organic larvicide

2. Observation on ethical considerations (Seeking approval from the Barangay LGU and

other authorized representatives to agencies concerned)

3. Collection of 10 larvae samples per experimental setups

4. Formulation of six concentrated solutions from the ingredients of the study. (Setup A:

2.5% concentration, Setup B: 5% concentration, Setup C: 10% concentration, Setup D: 25%

concentration, Setup E: 50% concentration, Setup F: 100% concentration)

Stage 2

1. Application of organic larvicide to the larvae samples

2. Application of Treatment (Larvicidal Assay)

3. Collection of Data

Stage 3

1. Presentation Analysis and Interpretation of Results from the data collected

2. Formulation of Research Output Base on the result of the study

Figure 1: Flow of the Study

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Larvicidal Assay

The following are to be undertaken in producing organic pesticide with the use of

Lemongrass (Cymbopogon Citratus), Garlic (Allium Sativum) and Papaya leaves (Carica papaya

L.). The three variables are to be chopped and crushed separately and the researchers will prepare

six concentrated solutions from the ingredients that is present in the study.

The following are the concentrated solutions that will be extracted from Lemongrass

(Cymbopogon Citratus), Garlic (Allium Sativum) and Papaya leaves (Carica papaya L.).

Setup A (2.5% concentration): 2.5ml of each extract; Lemongrass, Garlic, and Papaya

leaves extract & 67ml of water.

Setup B (5% concentration): 5ml of each extract; Lemongrass, Garlic, and Papaya leaves

extract & 60ml of water.

Setup C (10% concentration): 10ml of each extract; Lemongrass, Garlic, and Papaya

leaves extract & 45ml of water.

Setup D (25% concentration): 15ml of each extract; Lemongrass, Garlic, and Papaya

leaves extract & 30ml of water.

Setup E (50% concentration): 20ml of each extract; Lemongrass, Garlic, and Papaya

leaves extract & 15ml of water.

Setup F (100% concentration): 25ml of each extract; Lemongrass, Garlic, and Papaya

leaves extract & 0ml of water.

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 In the making of the organic larvicide, the researchers will prepare the ingredients needed

and the materials to be use. First, the 35 leaves of lemongrass will be rinsed off with great

caution. Next, it should be rinsed off again to ensure that there’s no dirt or grime left. Then, with

the use of knife, it should be chopped into fine pieces. After, the researchers will use mortar and

pestle to crush the lemongrass. To get its extract, the researchers will now put 15 tablespoons of

water to get the desired consistency. After, the process of extraction using a thin cloth will now

proceed, it will then be put into the six beakers with the proper measurements of its solutions

(Setup A: 2.5ml of lemongrass extract, Setup B: 5ml of lemongrass extract, Setup C: 10ml of

lemongrass extract, Setup D: 15ml of lemongrass extract, Setup E: 20ml of lemongrass extract,

Setup F: 25ml of lemongrass extract).

The next ingredient in the study is the garlic. The researchers will prepare 7 bulbs of

garlic. Using a knife and a cutting board, the garlic cloves must be chopped into small pieces.

After it is chopped, it will be crushed using mortar and pestle. After it is crushed, the researchers

will now add 15 tablespoons of water to effectively get its extract. Using a thin cloth, it will be

extracted, and the extracts will then be put and divided into the six beaker with different

measurements (Setup A: 2.5ml of garlic extract, Setup B: 5ml of garlic extract, Setup C: 10ml of

garlic extract, Setup D: 15ml of garlic extract, Setup E: 20ml of garlic extract, Setup F: 25ml of

garlic extract) joining the lemongrass extract.

The last ingredient in the study is the papaya leaves. The papaya leaves need to be freshly

picked from a fruit bearing papaya tree. The exact amount of papaya leaves is 3. After it is

picked, it will now be chopped using a knife and a cutting board. After we get the smallest pieces

it can get after being chopped, the researchers will now use mortar and pestle to crush the small

pieces of papaya leaves. The addition of 15 tablespoons of water will let the papaya leaves

14
to be

15
effectively extracted. The extracts will now be put into the six beakers with its different

measurements (Setup A: 2.5ml of papaya leaves extract, Setup B: 5ml of papaya leaves extract,

Setup C: 10ml of papaya leaves extract, Setup D: 15ml of papaya leaves extract, Setup E: 20ml

of papaya leaves extract, Setup F: 25ml of papaya leaves extract) combining with the lemongrass

extract and the garlic extract.

In addition to the concentrated solutions, different measurements of water will be put into

the six different beakers (Setup A: 67ml of water, Setup B: 60ml of water, Setup C: 45ml of

water, Setup D: 30ml of water, Setup E: 15ml of water, Setup F: 0ml of water) to test the lethal

effects of the three variables identified in the study.

Collection of Larvae Samples

The following will be the detailed steps in collecting the larvae samples in the different

experimental setups in conducting the study. The larvae samples will be collected near Luciano

B. Rama Sr. Memorial National High School in Barangay Esperanza, Poro, Cebu and it will be

collected from road swales near the school.

The number of larvae samples that will be collected from road swales near the school is

60 larvae in total. The collection of the larvae utilizes ladle and dipper to carefully place the

larvae samples in the container to be experimented. The researchers will transfer the larvae

samples into the six petri dishes. Each petri dish will contain 10 larvae samples to accomplish the

experiment.

After distributing the larvae samples into the six petri dishes, the application of six

concentrations will take part, conducting the study. The researchers will apply the measured

concentrations (Setup A: 2.5% concentration, Setup B: 5% concentration, Setup C: 10%

16
concentration, Setup D: 25% concentration, Setup E: 50% concentration, Setup F: 100%

concentration) by pouring each solution into their respective petri dish. Thus, conducting the

study.

Risk and Safety

The following are the identified risks that has the potential to occur in the study. Since the

entire study involves biological and environmental elements, risks are inevitable, and the study

needs to be carried out without any serious casualties. The following are the possible risks and

the safety precautions to prevent any serious damage and danger within the study.

Biohazard potential risks: The study primarily involves around the experimentation of

the ingredients that will be applied to the larvae samples. Thus, the contamination of the water

where the larvae reside can affect human health in the collection of the larvae samples. It is

highly recommended that the student-researchers take extra care in the collection of the larvae

samples by observing proper hygiene after the activity.

Chemical potential risks: In the formulation of the larvicide solutions, the ingredients

identified in the study can potentially harm the human skin due to the chemical components in

the ingredients. The student-researchers will wear safety attire like goggles and hand gloves to

avoid the risk of chemical hazard during and after the experimentation.

Utilization of Materials: The materials identified in the study can potentially harm the

researchers in conducting the study. This may involve in the chopping and crushing process of

the ingredients while conducting the study. The researchers will be oriented with the safety

measures on the conduct of the study to prevent harm as the experiment goes on.

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 Data Analysis

This study utilizes tables and graphs to present, analyze, and interpret the data collected

after the experimentation of the study. The tables below emphasize the elements and the proper

implementation that needs to be carried out in conducting the study.

Table 3: Distribution of Larvae Samples and Assignments of Concentrations

Experimental Number of Larvae Samples Concentration of Larvae

Setups Solution Applied

A 10 2.5%

B 10 5%

C 10 10%

D 10 25%

E 10 50%

F 10 100%

Table 3: Distribution of Larvae Samples and Assignments of Concentrations

Table 3 above will be use as a guide in the distribution of larvae and desired

concentrations for each setup. The table shows the exact concentration for each experimental

setups and the number of larvae samples that will coincide with the distribution of each

concentrated solutions in conducting the study.

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Table 4: Observation of Mosquito Larvae after the Application of Natural Larvicide

Number of Mosquito Larvae Died During the Different

Observations
Observation 1 Observation Observation Observation Observation
Experimental
2 3 4 5
Setups with level
(Application
of concentration
Time) (After 1 (After 5 (After (After 24
hour) hours) 10 hours)
hours)
A
2.5%
B
5%
C
10%
D
25%
E
50%
F
100%
Table 4: Observation of Mosquito Larvae after the Application of Natural Larvicide

Table 4 above will be used in the observation of the mosquito larvae after the application

of natural larvicide. This study observes and records the number of dead and alive mosquito

larvae. This table will also serve as a guide on the length of time that the larvicide’s

concentration is most effective with the different setups.

There will be five series of observations done to ensure the validity and reliability of the

result. This study compares the five series of observations to attain the most potent outcome of

this study. After the observations of this table, this study will acquire the most potent outcome by

getting the total population of dead mosquito larvae. This specifically answers the research

question number one, which is “How long will each concentration (2.5%, 5%, 10%, 25%, 50%

and 100%) make lethal effect on the collected larvae in each experimental setup?”

19
Table 5: Mortality Data of Mosquitos Larvae in each setup

Experimental Setups Mortality Rate

Table 5: Mortality Rate of Mosquito Larvae

Table 5 above will be used in presenting the mortality data of the mosquito larvae in each

setup. This table will utilize the total number of larvae that died taken from Table 4: Observation

of Mosquito Larvae after the Application of Natural Larvicide and calculate the percentage by

dividing the of the number of dead mosquito larvae to the total population per setup multiplied to

100 after the application of the natural larvicide. This table will help in determining the most

effective concentration in the study. This will serve as the basis in assessing the effectiveness of

each concentration in each experimental setup.

This study will further assess the total number of dead mosquito larvae in the study by

attaining the most effective concentration among the six experimental setups and the length of

time it will be observed in getting the maximum number of total mosquito larvae death in the

experimentation.

11
0
 To answer the research question number two, which is the “What is the mortality rate of

larvae of each experimental setup after the application of the six larvicide solutions with the

concentration of 2.5%, 5%, 10%, 25%, 50% and 100%?” this study will first assess the mortality

data from table 3.

After assessing the data, this study will now provide the mortality rate of the outcome of

each experimental setup by getting the percentage of the total population of dead larvae. This

will further serve as basis in identifying and answering the research question three, which is the

“Which among the six concentrations of natural mosquito Culicidae larvicide solutions made

from botanical extracts such as lemongrass Cymbopogon citratus, garlic Allium sativum and

papaya Carica papaya can kill the greatest number of mosquito larvae in the shortest period of

time?”

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