FM URD 006 Research Template 1 1 2 1 1 1 1

UNIVERSITY RESEARCH AND Document No.
: FM-URD-006
DEVELOPMENT CENTER Revision No.: 00
Effectivity Date: May 25, 2022
RESEARCH PROJECT Page 1 of 31
Combating Mosquito Menace: Unveiling the Potential of Lemongrass Oil

(Cymbopogon citratus), Oregano Oil (Origanum vulgare), and Lemon Fruit
(Citrus limon) as an Alternative Repellent Spray
A Research Proposal Presented

to the Faculty of the Senior High School
University of St. Louis
Tuguegarao City, Cagayan
In Partial Fulfillment
of the Requirements for the Program
SENIOR HIGH SCHOOL
By:
Andres, Xyzsa Heart S.
Buenaventura, Francheska A.
Fernandez, Jaymee Cassandra L.
Garcia, Khaleeya K.
Guaring, Abbie M.
October 2023
Combating Mosquito Menace: Unveiling the Potential of Lemongrass Oil (Cymbopogon citratus), Oregano Oil
(Origanum vulgare), and Lemon Fruit (Citrus limon) as an Alternative Repellent Spray | 1
UNIVERSITY RESEARCH AND Document No.: FM-URD-006
TABLE OF CONTENTS
Title Page
Title Page …….…………………………………………… 1
Dedication …….……………………………………………
Acknowledgement …….……………………………………………
Table of Contents ….……………………………………………… 2
List of Tables …………………………………………………
List of Figures …….…………………………………………… 3
Abstract …….……………………………………………
INTRODUCTION
Background of the Study ………………………………… 4
Research Questions ………………………………… 5
Significance of the Study ………………………………… 5
Literature Review ………………………………… 5
Research Paradigm ………………………………… 9
METHODS
Research Design ………………………………… 10
Locale of the Study ………………………………… 10
Materials of the Study ………………………………… 10
Instrument ………………………………… 14
Data Gathering Procedure ………………………………… 14
Data Analysis ………………………………… 18
RESULTS ……..………………..…………………
DISCUSSION ……..………………..…………………
CONCLUSION ……..………………..…………………
RECOMMENDATIONS ……..………………..…………………
REFERENCES ……..………………..………………… 19
APPENDICES
A. Literature Matrix ……..………………..…………...…….
B. Code Guide ………..………………..…………..………..
C. Data Matrix ………..………………..…………..………
D. Letter to the Vice President for Academics……………..
E. Letter to the Author of the Protocol….……….….......... 24
F. Letter to the Respondents ………..………………..…….
G. Informed Consent ………..………………..…………..….
H. Protocol ……..………………..…………….….……….. 15
CURRICULUM VITAE …….……………………………………
LIST OF FIGURES
Figure No. Title of Figure Page

1. Lemongrass Oil (Cymbopogon citratus), Oregano Oil
(Origanum vulgare), and Lemon fruit (Citrus limon) as a
repellent spray against Yellow Fever Mosquitoes (Aedes jjjjjjjjjsaegypti)………………….
…..………………..…………….…… 9
2. 60 cultured yellow fever mosquitoes (Aedes aegypti)….………. 10
3. Lemongrass oil…………………………………………………. 11
4. Oregano oil……………………………………………………. 11
5. Lemon fruit ………………………………………………………. 11
6. Mosquito nets …………………………………………………… 11
7. Clear cages …………………………………………………… 11
8. Clean containers (vials) ………………………………………… 12
9. Forceps/Tweezers ……………………………………………… 12
10. Notebook ……………………………………………………….. 12
11. Mortar and Pestle …………………………………………………. 12
12. Steam Distillation Apparatus …………………………………… 12
13. Pipettes ……………………………………………………………. 13
14. Clevenger Apparatus and Florence Flask ………………………… 13
15. Carrier oil (Coconut oil) …………………………………………. 13
16. Cheesecloth strainer ………………………………………..... 13
17. Clean Bottles …………………………………………………….. 13
18. Laboratory (fine type) Grater ……………………………………… 14
19. Graduated Cylinder ……………………………………………. 14
20. Glass Stirring Rod ………………………………………………. 14
INTRODUCTION
Mosquitoes, as per the Cambridge Dictionary, are tiny airborne insects that extract blood from
people and animals through bites. Certain mosquito species act as disease carriers, transmitting various
illnesses to humans. Recent approaches to disease control encompass vaccines, insecticides, repellents,
and improved waste management. Mosquito sprays are vital in communities, especially when dengue is
widespread. These can be used to keep mosquitoes away, preventing them from biting humans and
feeding on human blood, which can cause diseases such as dengue, malaria, West Nile virus, Zika virus,
and Chikungunya virus. Dengue is a viral infection that spreads from mosquitoes to people. It is more
common in tropical and subtropical climates (WHO, 2023), such as Kalinga, where marketed insect
repellents are difficult to access. In the Philippines, this is the most prevalent disease that can be gotten
from mosquitoes. It was reported that there were 226,497 cases of dengue fever in the Philippines in
2022, around 78,208 cases in 2021, and approximately 90,135 cases in 2020. (WHO, 2022). In addition to
that, in Tuguegarao City, Cagayan, the Provincial Epidemiology and Surveillance Unit (PESU) recorded
169 dengue cases from January to July, which is fewer than last year.The town of Santa Ana had the most
cases, mainly among children aged one to ten. (Cagayan Logs 169 Dengue Cases from January to July,
2023).
According to Dr. Mohamed E. I. Badawy, lead author and professor at the Department of
Pesticide Chemistry and Technology at Alexandria University in Egypt, essential oils from the peels of
citrus plants are highly efficient against larvae and adults. This suggests that there may be a natural
substance that is widely available that may be used to repel mosquitoes. For years, plant-based repellents
have been used as protection against mosquitoes. They were considered safe and toxic-free to be used by
people of all ages, unlike commercially bought mosquito repellents containing hazardous chemicals like
N, N-Diethyl-meta-toluamide, also called DEET. Although they are the most popular and effective
mosquito repellent, concerns have been raised about the possibility of unfavorable toxic consequences,
particularly in young children and pregnant women. Pyrethroids are highly toxic to aquatic organisms,
such as fish, insects, and crustaceans. They can cause acute and chronic toxicity, impair reproduction,
growth, and behavior, and affect food webs and ecosystem function (Werner & Moran, 2008).
Pyrethroids target the nervous system of insects and can also affect non-target organisms, including
humans. They can cause a range of neurological effects, such as tremors, convulsions, hyperactivity, and
impaired cognitive function, particularly in children. (Agnihotri, N., et al., 2020) .
Oregano extract and tea tree oil made the mosquito repellent essential oil effective and easy to use
and would last long due to the balanced amount of oregano extract and tea tree oil. (Casalla, S., et al.,
2021). The lemongrass leaf extract lotion was effective as an Aedes aegypti mosquito repellent (Ro'in, F.,
et al., 2020). After three trials with a two-hour interval, the researchers found that among the four-
treatment groups, 100% lemon extract concentration and commercial dog flea (100%) were the highest
percent mortality (Garnace, J. M., et al., 2019). Oils have different effects on pest insects. The most
important is that they block the air holes (spiracles) through which insects breathe, causing them to die
from asphyxiation. In some cases, oils also may act as poisons, interacting with the fatty acids of the
insect and interfering with normal metabolism. Oils also may disrupt how an insect feeds, a feature that is
particularly important in the transmission of some plant viruses by aphids. (Colorado State University
Extension, 2016). With dengue cases increasing rapidly higher each year with a proportional increase of
approximately 119% compared to 2021 (WHO, 2023), this research is to be proposed to have an effective
yet also a safe alternative to a mosquito repellent.
Research Questions
This study aims to determine the efficiency of Lemongrass Oil (Cymbopogon citratus), Oregano
Oil (Origanum vulgare), and Lemon Fruit (Citrus limon) as an alternative for commercial mosquito
sprays.
Specifically, it aims to answer the following questions:
1) What is the percentage of treatment of the Lemon Grass Oil (Cymbopogon citratus), Oregano Oil
(Origanum vulgare), and Lemon Fruit (Citrus limon)
a) 55% Lemongrass Oil, 40% Oregano Oil, and 5% Lemon Fruit
b) 40% Lemongrass Oil, 50% Oregano Oil, and 10% Lemon Fruit
c) 40% Lemongrass Oil, 55% Oregano Oil, and 5% Lemon Fruit
2) What is the effect of the different treatments formulated for mosquito repellency?
3) Is there a significant difference among the treatment with greater lemongrass oil percentage (a), the
treatment where the variables’ percentage are closest to each other (b), and the treatment with greater
oregano oil percentage (c)?
Research Hypothesis
There is no significant difference between the treatment with greater lemongrass oil percentage
(a), the treatment where the variables’ percentage are closest to each other (b), and the treatment with
greater oregano oil percentage (c).
Significance of the Study
This project aims to determine the efficiency of oregano oil, lemongrass oil, and lemon fruit as
an alternative mosquito repellent spray as it will contribute to the existing body of knowledge on the
effectiveness of organic mosquito repellants. Findings will be utilized to develop an effective alternative
to mosquito repellent by using organic materials to battle dengue and other mosquito-borne diseases.
This research will benefit the general public, more specifically, those who reside in rural places or
remote regions, like the mountainous provinces such as Kalinga, where marketed insect repellents are
difficult to access and will lead to a safer and better option for people, as the repellent spray can be
recreated and used against mosquitoes that are common in those areas. Furthermore, this study will be
environmentally friendly as the spray will not be as hazardous to those around it and can be used as a
much cheaper and safer alternative to other commercial products, where it will reduce the adverse effects
on the environment and human health. Future researchers interested in the same field can use this study
to improve further and expand.
Literature Review
This study is anchored on the concept of Dose-Response Relationship. The dose–response

relationship is the measurement of the relationship between the dose of a substance administered and its
overall effect (the response), either therapeutic or toxic. It is based on observed data from experimental
animal, human clinical, or cell studies. Generally, the higher the dose, the greater the response.
(Kilcoyne, A. (ed.), et al., 2013). It pertains to the impact an agent or exposure level has on an organism,
particularly in relation to the likelihood of a specific outcome occurring. It involves a scenario where
higher levels of exposure can lead to either an increased or decreased risk of that outcome. (Pettygrove,
S., 2016). The proposed concept is an accumulation of knowledge through time, and therefore, is not
attributed to only a single individual but also to the different researchers and scientists in various fields,
although, Paracelsus (1493-1541) must be recognized as the first one to realize that dose was the most
important issue to deal with in determining whether a chemical was toxic or not. (Waddell W. J., 2010).
The product will contain the three (3) main variables, namely the lemongrass oil, oregano oil, and lemon
fruit. These will be used to make three (3) different alternative mosquito sprays, where in each spray,
one variable will have a larger proportion of the concentration than the other two substances, and the
same goes for the other two formulations. Applying the concept, it suggests that as the concentration of
the selected variable/substance increases, the efficacy or repellency against the mosquitoes should also
change. This will allow a systematic examination of how various concentrations of the repellent spray
influence their efficacy in deterring mosquitoes enables the differentiation between concentration levels
and the identification of the most suitable concentration for attaining the intended level of mosquito
protection.
Mosquito Trends and Issues
Mosquitoes, as defined by the Cambridge Dictionary, are small flying insects that bite people
and animals to suck their blood. Some types of mosquitoes are known vectors of different diseases and
transmit them to humans. In recent years, traditional methods to combat these diseases have included
vaccines, insecticides, and repellents for prevention, as well as proper waste disposal.
Dengue fever, transmitted by Aedes aegypti mosquitoes, is a severe and sometimes deadly
disease, particularly prevalent in South and Southeast Asia. Globally, there are 390 million annual cases,
96 million showing symptoms and 294 million having no symptoms, with three Asian countries—
Vietnam, the Philippines, and Indonesia—reporting high numbers in 2022. Asia and the Pacific islands
account for 75% of worldwide dengue cases. Some developing countries use herbal remedies for
treatment. (Lubin, 2023). To address this ongoing public health concern, it's essential to focus on
controlling dengue vectors, understanding the disease, and developing vaccines and early diagnostic
tests. (Khan et al., 2023)
One of the commonly used as a prevention technique is to simply buy mosquito repellents and
other insecticides in the market. However, there are studies that establish a significant connection
between climate change, specifically humidity, and the mosquito population. (Beermann et al., 2023).
This growth exposes people to greater danger, especially since dengue cases in these areas are becoming
more common. Furthermore, other studies reveal a growing resistance to insecticides, specifically
towards their key components, pyrethroid and Dichlorodiphenyltrichloroethane (DDT). (Asgarian et al.,
2023).
Resistance to pesticides like dieldrin and DDT has emerged in mosquito populations across
several Asian countries. Thailand, despite banning DDT in agriculture since 1983, has reported
resistance due to its previous use in health and indoor malaria vector control. Malaysia has seen
resistance in both Aedes aegypti and Ae. albopictus, with the latter showing higher resistance, likely due
to its proximity to areas where dieldrin is still used in agriculture. India has recorded DDT resistance in
Ae. aegypti, with instances reported in various regions. Other countries, including China, Laos,
Myanmar, the Philippines, Vietnam, and Japan, have also documented varying degrees of resistance to
these pesticides in mosquito populations. (Asgarian et al., 2023). However, another study found no
evidence supporting epigenetic resistance in adult Ae. aegypti, suggesting insufficient activation of their
metabolic resistance pathways or the presence of unexplored epigenetic mechanisms. In disease vector
control, this could be positive news, as it implies one less avenue for mosquitoes to develop insecticide
resistance. This discovery raises hope for a potentially slower emergence of resistance within mosquito
populations, given the possibility of heritable cross-generational effects through the epigenome. (Parente,
2023).
In a study addressing humidity's impact on mosquito-borne diseases, it is emphasized that

maintaining the right body temperature and water balance is crucial for mosquitoes' health and
performance. Variations in humidity and temperature pose challenges for these insects, with higher
temperatures leading to increased water loss, especially at higher humidity fluctuations. (Brown et al.,
2023). Another study highlights how hotter and longer summers, changing precipitation patterns, and
increased outdoor activities can extend and intensify West Nile Virus (WNV) transmission in Germany.
The altered precipitation affects local mosquito populations transmitting WNV, while warmer summers
may reduce travel related WNV cases, favoring vacation destinations like Germany with fewer arbovirus
infections due to warm weather. (Asgarian et al., 2023). This issue is relevant to the Philippines, which
also faces climate change challenges.
Prevention of Mosquito-Borne Diseases
The World Health Organization (WHO) identified dengue as one of the top ten global health
threats in 2019. Sri Lanka grappled with a massive dengue epidemic in 2017, constituting the largest
outbreak in the country over the past three decades, with a staggering 186,101 reported cases and over
320 deaths. Dengue virus, transmitted by mosquito vectors like Aedes aegypti and Aedes albopictus,
was first serologically confirmed in Sri Lanka in 1962. The WHO introduced a "Global Strategy for
Dengue Prevention and `Control" spanning from 2012 to 2020, aiming to enhance dengue mortality and
morbidity outcomes by 2020 while estimating the actual disease burden. This study concludes that a
correlation exists between individuals possessing a strong understanding of the dengue burden and those
who are cognizant of patients' roles in dengue management. Moreover, there is a discernible upward
trend in awareness across all categories, especially among those with higher levels of education and
increased maturity by age, underscoring the significance of education and maturity in enhancing
awareness. (Jayawickreme et al., 2021)
The integrated approach to malaria prevention is gaining attraction as a strategy to combat

malaria. This approach advocates the simultaneous use of various malaria prevention methods in a
comprehensive manner. In the integrated approach, malaria prevention methods encompass those
providing individual and household protection (such as insecticide-treated nets (ITNs), insecticide
sprays, body mosquito repellents, IRS, and mosquito coils). It also includes strategies to reduce
mosquito breeding sites, involving actions like draining pools of water, larviciding, and clearing
unnecessary vegetation around homes. Additionally, the approach seeks to diminish the entry of
mosquitoes into houses through measures like installing mosquito screening in windows, ventilators,
and open eaves, along with closing windows and doors early in the evenings (Musoke et al., 2015). To
enhance the outcome, it is imperative to implement an action plan aimed at raising public awareness
regarding the role of the public and patients in dengue management. Furthermore, a research discovered
that participants displayed strong knowledge of and a high level of performance in mosquito breeding
site control (MBSC) practices. However, personal protection against mosquitoes, such as the use of
topical repellents, was perceived as relatively less effective and was practiced to a lesser extent
compared to MBSC practices, particularly larval source management. (Elsinga et al., 2017)
A study suggested that while the large-scale distribution of topical repellents may not yield a
community protective effect, targeting specific risk groups that potentially contribute disproportionately
to malaria transmission could prove more effective. Nonetheless, the effectiveness of introducing
standardized preventive measures without researching their local-level impact and without optimizing
community involvement strategies remains questionable (Gryseels et al., 2015). Another study
emphasizes that reducing the number of host-vector interactions constitutes an effective approach to
curbing the spread of vector-borne diseases. Currently, only a limited number of active ingredients
found in numerous commercially available formulations are widely employed to safeguard humans from
mosquitoes and other blood-sucking arthropods. (Moore et al., 2018)
The study of Herbuela et al., (2019) had assessed dengue fever (DF) knowledge, attitudes, and
practices (KAP) in pediatric (233 cases) and adult (17 cases) patients compared to community controls
in Metro Manila, Philippines. Both patient groups displayed lower practice scores than controls,
indicating a gap between knowledge/attitudes and preventive actions. In pediatric patients, attending
senior high school, longer hospital stays, and having a rash predicted better KAP. Protective factors
against DF included using mosquito-eating fish, having screened windows, and receiving the dengue
vaccine. This study underscores the importance of behavioral change and recommends implementing
the COMBI and HBM programs for effective DF prevention. (Herbuela et al., 2019).
Oregano Oil, Lemongrass Oil, and Lemon Fruit as Mosquito Repellants
Mosquitoes have been attributed to the transmission of several remarkably dangerous and deadly
diseases to people, including dengue fever, yellow fever, filariasis, and malaria. Commercially readily
available chemical-based insecticides and repellents have been shown to include dangerous and
poisonous substances that could endanger human health and be hazardous to non-target organisms.
However, concerns about its safety are still being taken into consideration.
The impact on aquatic life, human health, and the environment has all been examined. Because
of this, effective controls need to be developed to avoid these impacts. Safe control of health and
environmentally friendly green insecticides are intended to replace dangerous chemical-based

treatments with safe alternatives as a result of what appears to be a revived interest in natural product-
based repellents. (Ro'in et al., 2020)
It has long been known that Cymbopogon citratus (Lemon grass) oil works well as an insect
repellent; its success in keeping mosquitoes away has also been shown. The two main active ingredients
in lemongrass oil are geranial (α-citral) and neral (β-citral), and additional substances like citronellol
and geraniol, which are known repellents, are also present in small amounts. When used alone or in
combination, lemongrass essential oil has previously demonstrated a repellent effect against various
kinds of disease-transmitting mosquitoes. (Baldacchino et al., 2013)
Another variable that has been proven effective is the Oregano oil or Origanum vulgare, which is
considered as an excellent antiseptic and insect repellent. It has some active ingredients such as
carvacrol, thymol and α-terpinene reported being highly effective in repelling mosquitoes. (Sharififard
et al., 2018)
The phytochemical extracts from citrus fruit peels have proved effective as mosquito repellents
at reasonable concentrations. Today citrus essential oils as well as extracts from other plants such as
Cedar wood, Citronella, Eucalyptus, Pennyroyal, Turpentine, Winter green, have been identified as
particularly important natural resources of either pesticides or insecticides, or repellent. Phytochemical
extracts from citrus fruit peels have proved effective as mosquito repellents at reasonable
concentrations. (Effiom et al., 2012).
Process:
Research Paradigm 1. Obtain the variables:
 Oregano oil,
Lemongrass oil, and
Input:
Lemon fruit; Output:
55% Lemongrass oil
Mosquitoes
40% Oregano Oil
2. Collect the mosquitoes Three different
5% Lemon fruit
through larval sampling. formulations of
3. Extract oil from the mosquito
40% Lemongrass oil
lemongrass and oregano and sprays
50% Oregano Oil
grate the lemon fruit peel. consisting of
10% Lemon fruit
4. Formulation of the three the variables.
different repellent sprays.
40% Lemongrass oil
5. Testing the produced repellent
55% Oregano Oil
sprays on the cultured
5% Lemon fruit
mosquitoes
NEGATIVE CONTROL
6. Collect data based on the
(Mosquitoes)
results.
7. Interpretation and data
analysis
Figure 1. Lemongrass Oil (Cymbopogon citratus), Oregano Oil (Origanum vulgare), and Lemon fruit
(Citrus limon) as a repellent spray against Yellow Fever Mosquitoes (Aedes aegypti)
Figure 1 illustrates the processes and concentrations employed for testing the repellency of
Lemongrass oil, oregano oil, and lemon fruit. The figure commences with the collection of sixty (60)
cultured mosquitoes. Next, it involves the extraction of oil from lemongrass and oregano, as well as the
grating of the lemon fruit. The third step encompasses the execution of the experiment, followed by data
analysis and interpretation.
METHODS
Research Design
This study will utilize a quantitative experimental type of research to determine the optimal
concentrations of Lemongrass oil (Cymbopogon citratus), Oregano oil (Origanum vulgare), and Lemon
fruit (Citrus limon) against mosquitoes, specifically, yellow fever Mosquitoes (Aedes aegypti). Through
systematic variation of these variables, the goal is to identify the most effective formulation for reducing
mosquito populations. The study involves the application of these formulations to mosquitoes, assessing
their efficiency in exterminating the sample population.
Locale of the Study
The study will be conducted at Cagayan State University - Andrews Campus, as their laboratory
provides sufficient materials and equipment needed for the procedures to be performed.
Materials of the Study
Lemongrass Oil (Cymbopogon citratus), Oregano Oil (Origanum vulgare), Lemon Fruit (Citrus
limon), and Cultured Mosquitoes (Aedes aegypti) are the subjects of this study. The sample population
of mosquitoes to be used will be derived from the gathered larvae (larval sampling) which will be
divided into three groups for the testing, as there will be a total of 60 mosquitoes (20 for each
formulation) while the lemongrass oil, oregano oil, and lemon fruit will be obtained from a reputable
source.
Figure 2. 60 cultured yellow fever mosquitoes (Aedes aegypti)

The mosquitoes will be used as the negative control of our study.
Figure 3. Lemongrass oil (Cymbopogon citratus)

The lemongrass oil will be used as an ingredient to make experimental repellent sprays.
Figure 4. Oregano oil (Origanum vulgare)

The oregano oil will be used as an ingredient to make experimental repellent sprays.
Figure 5. Lemon fruit (Citrus limon)
The lemon fruit will be used as an ingredient to make experimental repellent sprays.
Figure 6. Mosquito net/s

This will be used to catch the cultured mosquitoes from the collection site.
Figure 7. Clear cage/s

Clear cages will be used to contain the collected mosquitoes.
Figure 8. Clean containers (vials)

Vials will be used for the formulation of the repellent sprays.
Figure 9. Forceps/Tweezers
This will be used to grab onto small objects that are too small for our hands.
Figure 10. Notebook

This will be used for data recording.
Figure 11. Mortar and Pestle

This will be used to grind the leaves of the oregano and lemongrass as a step to obtain their oil.
Figure 12. Steam Distillation Apparatus

This will be used to obtain the oil from the lemongrass and oregano.
Figure 13. Pipette/s

Pipettes will be used to measure the correct amount of the ingredients.
Figure 14. Clevenger Apparatus and Florence Flask

These will be used to obtain the oil from the lemongrass and oregano
Figure 15. Carrier oil (Coconut oil)

This oil will be used to carry the natural components from the oregano and lemongrass to turn it into oil.
Figure 16. Cheesecloth Strainer

Strainers will be used to remove any other material from the lemongrass oil and oregano oil.
Figure 17. Clean Bottles

These will be used for oil storage.
Figure 18. Laboratory (Fine type) Grater

This will be used to grate the lemon fruit.
Figure 19. Graduated Cylinder

This will be used to correctly measure the amount of ingredients for the formulation of the repellents.
Figure 20. Glass Stirring Rod

This will be used to stir the ingredients evenly.
Research Instruments
The researchers will use the protocol of Medlock et al., (2018) for the collection of mosquitoes,
the study of Wagh et al., (2021) for the extraction of lemongrass oil, the study of Chen et al., (2020) for
the extraction of oregano oil, the protocol of Henderson et al., (2018) for the proper grating of lemon
peel, and the protocol of Food and Agriculture Organization (FAO)/International Atomic Energy Agency
(IAEA), (2023) for the proper disposal of the mosquitoes.
Data Gathering Procedures
Before starting, the researchers will need to obtain permission from the authors of the protocols to
be used. First, the three substances, namely Lemongrass Oil (Cymbopogon citratus), Oregano Oil
(Origanum vulgare), and Lemon Fruit (Citrus limon), will be obtained in pure form from a reliable
source, and the mosquitoes to be used in the experiment will be gathered through larval sampling from
breeding sites. Second, is to set all needed materials by extracting oil from the lemongrass and oregano
through steam distillation and grating the lemon peel. The next step would be to formulate three different
repellent sprays using each substance as the main ingredient, with different concentrations in all three
formulations. The spray formulations will then be tested for efficacy, with 15 sprays per set. The testing
will be conducted in a laboratory setting with a cage containing mosquitoes. The products will be used
against mosquitoes. This process will be repeated multiple times to obtain a large enough sample size for
statistical analysis.
Protocol
A. Clinical Laboratory Testing for Sensitization and Allergies

1. Observe Sensitization and Allergies
a. Be aware of any known allergies or sensitivities you may have to specific
essential oils or particular plants.
B. Collection of Mosquitoes (Aedes aegypti) (Medlock et al., 2018)

1. Identify the Collection Site
a. Select a suitable collection site, such as an area with a high mosquito
population, near stagnant water sources, or near vegetation.
2. Obtain Permission
a. If the collection site is private property or requires permission, obtain the
necessary approvals from relevant authorities or property owners.
3. Choose Collection Method

a. Larval sampling from breeding sites will be used as the collection
method of mosquitoes.
4. Implement Collection Procedure

a. Carry out the collection process at the selected site, following the chosen
method and adhering to safety precautions.
b. Capture mosquitoes using nets.
c. Record the collection date, time, location, and relevant observations in
the notebook.
5. Preserve and Transport Mosquitoes

a. Transfer captured mosquitoes into labeled containers, ensuring proper
identification and separation by collection site, date, or species.
b. Add a desiccant (silica gel or paper) to control moisture and preserve
specimens.
c. Transport the collected mosquitoes in a cool, dry, and secure manner to
the laboratory for further analysis.
C. Extracting Oil from Lemongrass (Wagh et al., 2021)

1. Gather Lemongrass
a. Obtain fresh lemongrass stalks from a reputable source. Ideally, choose organic
lemongrass for the best quality oil.
2. Cut and Crush Lemongrass
a. Trim the lemongrass stalks and discard the upper leaves. Cut the stalks into
smaller pieces for easier processing.
b. Crush the lemongrass using a mortar and pestle, or blend it until it forms a
coarse paste. Crushing helps release the essential oil.
3. Distillation Process
a. Set up the distillation apparatus according to the manufacturer's
instructions or DIY setup.
b. Place the crushed lemongrass in the distillation chamber or a heat-
resistant container above a heat source (if using a DIY setup).
c. Add water to the bottom of the distillation apparatus or the pot in your DIY
setup.
d. Heat the water and let the steam pass through the lemongrass, carrying the
essential oil.
4. Collect the Condensed Oil

a. As the steam passes through the lemongrass, it carries the essential oil. The
steam then condenses in a separate chamber or a condenser coil.
b. Collect the condensed liquid, which consists of water and the essential oil. This
liquid is known as hydrosol or essential oil-infused water.
5. Separate the Oil

a. The essential oil will float on top of the hydrosol. Carefully decant or
separate the oil from the water using a pipette.
b. Transfer the extracted lemongrass oil into a clean container for storage.
6. Store the Oil

a. Store the lemongrass oil in a cool, dark place away from direct sunlight and
heat. Ensure the bottles are tightly sealed to preserve the oil's quality and
aroma.
D. Extracting Oil from Oregano (Chen et al., 2020)

1. Gather Oregano
a. Obtain fresh oregano leaves from a reputable source. Ideally, choose
organic oregano for the best quality oil.
2. Crush Oregano Leaves

a. Wash and thoroughly dry the oregano leaves.
b. Using the mortar and pestle, crush the leaves to release the essential oil.
3. Infuse the Oil

a. Put the crushed oregano leaves in the Clevenger apparatus or Florence flask,
adding about a teaspoon or a carrier oil of your choice. Heat the mixture for 2 to
4 hours using steam distillation, depending on the sample size (50g-100g).
4. Strain the Oil
a. Once infused, remove it from the heat and let it cool slightly.
b. Place a cheesecloth or a fine-mesh strainer over a clean glass jar or bottle.
c. Pour the infused oil through the strainer to remove any plant material,
ensuring only the oil passes through.
5. Store the Oil

a. Transfer the strained oil into clean, dark glass bottles or containers for
storage.
b. Store the oregano oil in a cool, dark place away from direct sunlight and heat
to preserve its quality and potency.
E. Grating of Lemon Peel (Henderson et al., 2018)

1. Gather Lemon
a. Prepare and wash the lemons.
2. Grate Lemon Peel

a. Grate lemon using a laboratory or fine-type kitchen grater.
3. Store or Use the Grated Lemon Peel

a. If the grated peel will not be used immediately, store it in an airtight
container in the refrigerator.
b. Use the grated lemon peel according to the specific amount of the E.
F. Combining Lemongrass Oil (Cymbopogon citratus), Oregano Oil (Origanum vulgare), and
Lemon Fruit (Citrus limon) to formulate the repellent
1. Treatment A (55% Lemongrass Oil, 40% Oregano Oil, and 5% Lemon Fruit)
a. On a 100ml graduated cylinder, pour in 33ml of extracted lemongrass oil, 24ml
of extracted oregano oil, and 3g of grated lemon peel.
b. Gently stir the solution with a glass stirring rod.
c. Store the solution in a sterile spray bottle.
2. Treatment B (40% Lemongrass Oil, 50% Oregano Oil, and 10% Lemon Fruit)
3. Treatment C (40% Lemongrass Oil, 55% Oregano Oil, and 5% Lemon Fruit)
G. Proper Disposing of Mosquitoes (American Biological Safety Association, 2017)

1. Anesthetize the mosquitoes
a. Since live mosquitoes are involved, they may need to be anesthetized before
disposal to ensure they are no longer active.
2. Contain and secure the mosquitoes

a. Once the mosquitoes are no longer active, it is crucial to contain them to
prevent their escape. Use appropriate containers, such as sealed bags or
containers with secure lids, to ensure containment.
3. Sterilize the containers

a. To minimize the risk of accidental release or contamination, sterilize the
containers used for mosquito disposal. This can be done by wiping them with a
suitable disinfectant or autoclaving them, depending on the specific

requirements.
4. Dispose of the mosquitoes

a. Proper disposal methods will depend on local regulations and institutional
guidelines. In general, the mosquitoes should be disposed of to prevent their
escape and minimize any potential risks. This may include incineration, deep
burial, or other approved methods.
Data Analysis
To analyze the results, a one-way ANOVA (Analysis of Variance) test will be conducted to
determine if there is a significant difference in the number of mosquito deaths in each of the three
repellent sprays. Post hoc testing will determine which specific repellent spray is the most effective if a
significant difference is found.
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Insecticide Resistance of Aedes aegypti and Ae. albopictus, Vectors of Arboviruses of
Chikungunya, Dengue, Zika and Yellow Fever. Journal of Arthropod-borne Diseases.
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Badawy, M. E. I., Taktak, N. E. M., & El-Aswad, A. F. (2017). Chemical composition of the essential
oils isolated from peel of three citrus species and their mosquitocidal activity against Culex
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Baldacchino, F., Tramut, C., Salem, A., Liénard, E., Delétré, E., Franc, M., Martin, T., Duvallet, G., &
Jay‐Robert, P. (2013). The repellency of lemongrass oil against stable flies, tested using video
tracking. Parasite, 20, 21. https://doi.org/10.1051/parasite/2013021
Beermann, S., Dobler, G., Faber, M., Frank, C., Habedank, B., Hagedorn, P., Kampen, H., Kuhn, C.,
Nygren, T., Schmidt‐Chanasit, J., Schmolz, E., Stark, K., Ulrich, R. G., Weiß, S., & Wilking, H.
(2023). Impact of climate change on vector- and rodent-borne infectious diseases. PubMed,
8(Suppl 3), 33–61. https://doi.org/10.25646/11401
Brown, J. J., Pascual, M., Wimberly, M. C., Johnson, L. R., & Murdock, C. C. (2023). Humidity – The
overlooked variable in the thermal biology of mosquito ‐borne disease. Ecology Letters, 26(7),
1029–1049. https://doi.org/10.1111/ele.14228
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Chen, L., Cör, D., Simonovska, J., Knez, Ž., Kavrakovski, Z., & Rafajlovska, V. (2020). Extraction
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Olszewska−Słonina, D. (2018). Current research on the safety of pyrethroids used as insecticides.
Medicina-lithuania, 54(4), 61. https://doi.org/10.3390/medicina54040061
Crisis. (2023, March 6). Philippines: Elevated dengue fever activity reported nationwide through
February. Philippines: Elevated Dengue Fever Activity Reported Nationwide Through February |
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Colorado State University Extension. (2016, May 19). Insect Control: Horticultural Oils - 5.569 -
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Elsinga, J., Van Der Veen, H. T., Gerstenbluth, I., Burgerhof, J. G. M., Dijkstra, A., Grobusch, M. P.,
Tami, A., & Bailey, A. (2017). Community participation in mosquito breeding site control: an
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[IAEA]. (2023). GUIDELINES FOR BIOSAFETY AND BIOSECURITY IN MOSQUITO
REARING FACILITIES. International Atomic Energy Agency.
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Gerrets, R., Sochantha, T., Coosemans, M., & Grietens, K. P. (2015). Factors influencing the use
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Against Escherichia coli. © 2012-2021 American Academic Scientific Research Journal for
Engineering, Technology, and Sciences.
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Herbuela, V. R. D. M., De Guzman, F. S., Sobrepeña, G. D., Claudio, A. B. F., Tomas, A. C. V., Reyes,
C. M. A., Regalado, R. A., Teodoro, M. M., & Watanabe, K. (2019). Knowledge, Attitude, and
Practices Regarding Dengue Fever among Pediatric and Adult In-Patients in Metro Manila,
Philippines. International Journal of Environmental Research and Public Health, 16(23), 4705.
https://doi.org/10.3390/ijerph16234705
Jayawickreme, K. P., Jayaweera, D. K., Weerasinghe, S., Warapitiya, D. S., & Subasinghe, S. (2021). A
study on knowledge, attitudes and practices regarding dengue fever, its prevention and
management among dengue patients presenting to a tertiary care hospital in Sri Lanka. BMC
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Wang, S. (2023). Dengue Overview: An updated systemic review. Journal of Infection and
Public Health, 16(10), 1625–1642. https://doi.org/10.1016/j.jiph.2023.08.001
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https://scholarsarchive.byu.edu/ballardbrief/vol2023/iss1/7/
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for mosquitoes, sandflies, biting midges and ticks. EFSA Supporting Publications, 15(6).
https://doi.org/10.2903/sp.efsa.2018.en-1435
Moore, E., Scott, M. A., Rodriguez, S. D., Mitra, S., Vulcan, J., Cordova, J. J., Chung, H., De Souza, D.
L. L., Gonzales, K. K., & Hansen, I. A. (2018). An online survey of personal mosquito-repellent
strategies. PeerJ, 6, e5151. https://doi.org/10.7717/peerj.5151
Parente, M. E., & Stoddard, P. K. (2023). Inducible Resistance to Pyrethroid Insecticide is Lacking in
Adult Aedes aegypti Mosquitoes. FIU Undergraduate Research Journal, 1(1).
https://doi.org/10.25148/urj.010322
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Assessment. Encyclopedia Britannica. https://www.britannica.com/science/dose-response-
relationship
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EXTRACT LOTION (Cymbopogon nardus) AS REPELLENT OF AEDES AEGYPTI
MOSQUITOES. Bulletin Kesehatan Lingkungan Dan Masyarakat, 39(4), 164–170.
https://doi.org/10.31983/keslingmas.v39i4.6578
Sharififard, M. (2018, December 1). Chemical Composition and Repellency of Origanum vulgare
Essential Oil against Cimex lectularius under Laboratory Conditions. PubMed Central (PMC).
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423461/
Waddell, W. J. (2010). History of dose response. Journal of Toxicological Sciences, 35(1), 1–8.
https://doi.org/10.2131/jts.35.1
Wagh, A. M., Jaiswal, S., & Bornare, D. T. (2021). A review: Extraction of essential oil from lemon grass
as a preservative for animal products. Journal of Pharmacognosy and Phytochemistry, 10(3S),
26–31. https://doi.org/10.22271/phyto.2021.v10.i3sa.14187
Werner, I., & Moran, K. D. (2008). Effects of pyrethroid insecticides on aquatic organisms. In Acs
Symposium Series (pp. 310–334). https://doi.org/10.1021/bk-2008-0991.ch014
WHO & World Health Organization [WHO]. (2023, March 23). Disease outbreak news.
https://www.who.int/emergencies/disease-outbreak-news/2023-DON448
World Health Organization (WHO). (2019). Ten threats to global health in 2019.
https://www.who.int/news-room/spotlight/ten-threats-to-global-health-in-2019
World Health Organization: WHO & World Health Organization: WHO. (2023). Dengue and severe
dengue. www.who.int. https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-
dengue
LETTER TO THE AUTHOR OF THE PROTOCOL
October 26, 2023
ALHOI HENDRY HENDERSON

Faculty of Medicine, Universitas Prima Indonesia, Medan, North Sumatera, Indonesia
Dear Sir:
Greetings of Peace!
We, the Grade 12 STEM-Health students of University of Saint Louis Tuguegarao, Cagayan, Philippines
will be conducting a study entitled “Combating Mosquito Menace: Unveiling the Potential of
Lemongrass Oil (Cymbopogon citratus), Oregano Oil (Origanum vulgare), and Lemon Fruit
(Citrus limon) as an Alternative Repellent Spray” as a final requirement for our subjects, Practical
Research 2 and Research Project. We are writing to ask written permission to use the protocol from
"Antimicrobial Activity of Lemon (Citrus limon) Peel Extract Against Escherichia coli" in our research
study. Our research study utilizes a Quantitative Research Design with lemongrass oil, oregano oil, and
lemon fruit as materials against yellow fever mosquitoes. This research study aims to determine the
efficacy level of the said materials to know which treatment/concentration would be the best against
mosquitoes (Aedes aegypti). Our research is being supervised by our teacher, Mr. Darwin Dumlao,
MST.
We, the researchers, are asking for permission to adapt the protocol and make necessary modifications in
order to make it more relevant in our study. We plan to employ the protocol in the process of the
experiment, specifically during the process of acquiring the needed amount of lemon fruit peel. Through
the use of the adapted and modified protocol, we will be able to acquire the right amount of the said
material. Subsequently, the lemon fruit will be combined with the other ingredients in the correct
concentration levels. The collected data will be processed through a one-way ANOVA test. The objective
of our study is to determine the efficiency of oregano oil, lemongrass oil, and lemon fruit as an alternative
mosquito repellent spray as it will contribute to the existing body of knowledge on the effectiveness of
organic mosquito repellants. We would like to use your protocol under the following conditions:
 We will use the protocol only for our research study and will not sell or use it for any other
purposes.
 We will include a statement of attribution and copyright on all copies of the instrument. If you
have a specific statement of attribution that you would like for us to include, please provide it in
your response.
 At your request, we will send a copy of our completed research study to you upon completion of
the study and/or provide a hyperlink to the final manuscript.
If these are acceptable terms and conditions, please indicate so by replying to us through e-mail at
khaleeyagharzeeya06@gmail.com.
Sincerely,
XYZSA HEART S. ANDRES FRANCHESKA A. BUENAVENTURA
ABBIE M. GUARING JAYMEE CASSANDRA L. FERNANDEZ
KHALEEYA K. GARCIA
Researchers
October 26, 2023
AKASH WAGH
Society of Pharmacognosy and Phytochemistry,
#3, Pocket H-34, Sector-3, Rohini, New Delhi, India- 110085
Dear Sir:
Greetings of Peace!
We, the Grade 12 STEM-Health students of University of Saint Louis Tuguegarao, Cagayan,
Philippines will be conducting a study entitled “Combating Mosquito Menace: Unveiling the
Potential of Lemongrass Oil (Cymbopogon citratus), Oregano Oil (Origanum vulgare), and
Lemon Fruit (Citrus limon) as an Alternative Repellent Spray” as a final requirement for our
subjects, Practical Research 2 and Research Project. We are writing to ask written permission to use
the protocol from "A review: Extraction of essential oil from lemon grass as a preservative for animal
products" in our research study. Our research study utilizes a Quantitative Research Design with
lemongrass oil, oregano oil, and lemon fruit as materials against yellow fever mosquitoes. This
research study aims to determine the efficacy level of the said materials to know which
treatment/concentration would be the best against mosquitoes (Aedes aegypti). Our research is being
supervised by our teacher, Mr. Darwin Dumlao, MST.
We, the researchers, are asking for permission to adapt the protocol and make necessary modifications
in order to make it more relevant in our study. We plan to employ the protocol in the process of the
experiment, specifically during the process of acquiring the needed amount of lemongrass oil.
Through the use of the adapted and modified protocol, we will be able to acquire the right amount of
the said material. Subsequently, the lemongrass oil will be combined with the other ingredients in the
correct concentration levels. The collected data will be processed through a one-way ANOVA test.
The objective of our study is to determine the efficiency of oregano oil, lemongrass oil, and lemon
fruit as an alternative mosquito repellent spray as it will contribute to the existing body of knowledge
on the effectiveness of organic mosquito repellants. We would like to use your protocol under the
following conditions:
• We will use the protocol only for our research study and will not sell or use it for any other
purposes.
• We will include a statement of attribution and copyright on all copies of the instrument. If you
your response.
• At your request, we will send a copy of our completed research study to you upon completion of
Sincerely,
KHALEEYA K. GARCIA
Researchers
October 26, 2023
Dr. JOLYON MEDLOCK

Public Health England
Dear Sir:
Greetings of Peace!
the protocol from "Field sampling methods for mosquitoes, sandflies, biting midges and ticks" in our
research study. Our research study utilizes a Quantitative Research Design with lemongrass oil,
oregano oil, and lemon fruit as materials against yellow fever mosquitoes. This research study aims to
determine the efficacy level of the said materials to know which treatment/concentration would be the
best against mosquitoes (Aedes aegypti). Our research is being supervised by our teacher, Mr. Darwin
Dumlao, MST.
experiment, specifically during the process of collecting the correct number of mosquitoes to be used
in the experiment. Through the use of the adapted and modified protocol, we will be able to acquire
the exact number of subjects guided by your paper. Subsequently, the mosquitoes collected will be
used to test which of the treatments/formulations would be the most efficient. The collected data will
be processed through a one-way ANOVA test. The objective of our study is to determine the
efficiency of oregano oil, lemongrass oil, and lemon fruit as an alternative mosquito repellent spray as
it will contribute to the existing body of knowledge on the effectiveness of organic mosquito
repellants. We would like to use your protocol under the following conditions:
● We will use the protocol only for our research study and will not sell or use it for any other
purposes.
● We will include a statement of attribution and copyright on all copies of the instrument. If you
your response.
● At your request, we will send a copy of our completed research study to you upon completion of
Sincerely,
KHALEEYA K. GARCIA
Researchers
October 26, 2023
Dr. MASA KNEZ MAREVCI

Professor (Faculty of chemistry and chemical engineering) Employment
University of Maribor: Maribor, SI
Dear Ma'am:
Greetings of Peace!
the protocol from "Extraction Techniques and Analytical Methods for Characterization of Active
Compounds in Origanum Species" in our research study. Our research study utilizes a Quantitative
Research Design with lemongrass oil, oregano oil, and lemon fruit as materials against yellow fever
mosquitoes. This research study aims to determine the efficacy level of the said materials to know
which treatment/concentration would be the best against mosquitoes (Aedes aegypti). Our research is
being supervised by our teacher, Mr. Darwin Dumlao, MST.
experiment, specifically during the process of acquiring the needed amount of oregano oil. Through
the use of the adapted and modified protocol, we will be able to extract the correct amount of oil from
the oregano. Subsequently, the oregano oil will be combined with the other ingredients at the correct
concentration levels. The collected data will be processed through a one-way ANOVA test. The
objective of our study is to determine the efficiency of oregano oil, lemongrass oil, and lemon fruit as
an alternative mosquito repellent spray as it will contribute to the existing body of knowledge on the
effectiveness of organic mosquito repellants. We would like to use your protocol under the following
conditions:
● We will use the protocol only for our research study and will not sell or use it for any other
purposes.
● We will include a statement of attribution and copyright on all copies of the instrument. If you
your response.
● At your request, we will send a copy of our completed research study to you upon completion of
Sincerely,
KHALEEYA K. GARCIA
Researchers
October 26, 2023
FOOD AND AGRICULTURE ORGANIZATION

Food and Agriculture Organization of the United Nations/International Atomic Energy Agency,
Vienna, Austria
Dear Ma’am/Sir:
Greetings of Peace!
the protocol from "Guidelines for Biosafety and Biosecurity in Mosquito Rearing Facilities" in our
research study. Our research study utilizes a Quantitative Research Design with lemongrass oil,
oregano oil, and lemon fruit as materials against yellow fever mosquitoes. This research study aims to
determine the efficacy level of the said materials to know which treatment/concentration would be the
best against mosquitoes (Aedes aegypti). Our research is being supervised by our teacher, Mr. Darwin
Dumlao, MST.
experiment, specifically after conducting the experiment, where the mosquitoes will be disposed of
properly. Through the use of the adapted and modified protocol, we will be able to safely finish our
data gathering procedure guided by your protocol. The collected data will be processed through a one-
way ANOVA test. The objective of our study is to determine the efficiency of oregano oil, lemongrass
oil, and lemon fruit as an alternative mosquito repellent spray as it will contribute to the existing body
of knowledge on the effectiveness of organic mosquito repellants. We would like to use your protocol
under the following conditions:
 We will use the protocol only for our research study and will not sell or use it for any other
purposes.
 We will include a statement of attribution and copyright on all copies of the instrument. If you
your response.
 At your request, we will send a copy of our completed research study to you upon completion of
Sincerely,
KHALEEYA K. GARCIA
Researchers

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UNIVERSITY RESEARCH AND Document No.

Combating Mosquito Menace: Unveiling the Potential of Lemongrass Oil

A Research Proposal Presented

Figure No. Title of Figure Page

Significance of the Study

This study is anchored on the concept of Dose-Response Relationship. The dose–response

Mosquito Trends and Issues

In a study addressing humidity's impact on mosquito-borne diseases, it is emphasized that

Prevention of Mosquito-Borne Diseases

The integrated approach to malaria prevention is gaining attraction as a strategy to combat

Oregano Oil, Lemongrass Oil, and Lemon Fruit as Mosquito Repellants

environmentally friendly green insecticides are intended to replace dangerous chemical-based

Locale of the Study

Materials of the Study

Figure 2. 60 cultured yellow fever mosquitoes (Aedes aegypti)

Figure 3. Lemongrass oil (Cymbopogon citratus)

Figure 4. Oregano oil (Origanum vulgare)

Figure 6. Mosquito net/s

Figure 7. Clear cage/s

Figure 8. Clean containers (vials)

Figure 10. Notebook

Figure 11. Mortar and Pestle

Figure 12. Steam Distillation Apparatus

Figure 13. Pipette/s

Figure 14. Clevenger Apparatus and Florence Flask

Figure 15. Carrier oil (Coconut oil)

Figure 16. Cheesecloth Strainer

Figure 17. Clean Bottles

Figure 18. Laboratory (Fine type) Grater

Figure 19. Graduated Cylinder

Figure 20. Glass Stirring Rod

Data Gathering Procedures

A. Clinical Laboratory Testing for Sensitization and Allergies

B. Collection of Mosquitoes (Aedes aegypti) (Medlock et al., 2018)

3. Choose Collection Method

4. Implement Collection Procedure

5. Preserve and Transport Mosquitoes

C. Extracting Oil from Lemongrass (Wagh et al., 2021)

4. Collect the Condensed Oil

5. Separate the Oil

6. Store the Oil

D. Extracting Oil from Oregano (Chen et al., 2020)

2. Crush Oregano Leaves

3. Infuse the Oil

5. Store the Oil

E. Grating of Lemon Peel (Henderson et al., 2018)

2. Grate Lemon Peel

3. Store or Use the Grated Lemon Peel

G. Proper Disposing of Mosquitoes (American Biological Safety Association, 2017)

2. Contain and secure the mosquitoes

3. Sterilize the containers

suitable disinfectant or autoclaving them, depending on the specific

4. Dispose of the mosquitoes

LETTER TO THE AUTHOR OF THE PROTOCOL

October 26, 2023

ALHOI HENDRY HENDERSON

XYZSA HEART S. ANDRES FRANCHESKA A. BUENAVENTURA

ABBIE M. GUARING JAYMEE CASSANDRA L. FERNANDEZ

LETTER TO THE AUTHOR OF THE PROTOCOL

October 26, 2023

XYZSA HEART S. ANDRES FRANCHESKA A. BUENAVENTURA

ABBIE M. GUARING JAYMEE CASSANDRA L. FERNANDEZ

LETTER TO THE AUTHOR OF THE PROTOCOL