ABSTRACT

Keywords: Oregano (Origanum vulgare), Larvicide, Efficacy, Aedis aegypti

This study aims to investigate the larvicidal efficacy of Oregano (Origanum

vulgare) powdered dried leaves against dengue-carrying mosquitoes, Aedis aegypti,
that have continually devastating and killed humans for several decades now.
Eliminating the source of infection is an essential step in controlling these vectors but
indiscriminate application of commercial larvicide will just lead to physiological
resistance on the part of these little assassins and will also result to a long-term effect
on non-target organisms and other environmental components. Therefore, the use of
commercial larvicide is undesirable, so this study will answer to the urgent call for
economical, eco-friendly yet effective alternative source of larvicide to control mosquito
menace.

This is an experimental research in two groups, Experimental and Control, and

each group has three replicates. Each replicate has 10 second instar larvae of Aedis
aegypti. The Experimental group is treated with the powdered dried leaves of the
Oregano (Origanum vulgare) while the control group is treated with the commercial
larvicide, Antimos 1. Based on the gathered data, in Replicate 1, Oregano (Origanum
vulgare) powdered dried leaves killed 7 out of 10 second instar larvae of the Aedis
aegypti while in Replicate 2, there were 8 larvae died after the treatment and another 7
larvae killed in the 3rd replicate. On the other hand, commercial larvicide, Antimos 1,
also showed remarkable performance after killing 9 larvae in Replicate 1 and 8 larvae in
Replicate 2 and 3. The results denote that the commercial larvicide, Antimos 1, is very
effective in killing Aedis aegypti larvae but Oregano (Origanum vulgare) powdered dried
leaves is also an effective larvicide after killing more than half of the larvae in each
group per replicate and therefore, could be an alternative source of larvicide which is
safe, eco-friendly, and very accessible for the community to use.
 RATIONALE

The world has been continually devastated by tiny creatures yet

considered as the greatest human assassins for they have the potential and lethal

capacity to kill more than one million victims a year around the globe. Mosquito borne

diseases such as Yellow Fever, Zika infection, Dengue and Chikungunya are consistently

the major health problems for the people. Dengue alone has infected about 50 - 100

million people, more than 500, 000 of them develop into dengue hemorrhagic fever, a

more severe form of the disease and results in up to approximately 22, 000 deaths

annually worldwide according to World Health Organization (WHO).

Philippines being located in the tropical zone, has not escaped from these

vectors. Based on the latest report on dengue cases early this year 2018, there were

15, 599 recorded by the Department of Health (DOH) which is actually lower as

compared to last year’s record on dengue cases at the same period which has reached

23, 131 cases. Though this year’s record on dengue cases has dropped or is lower than

the previous year, it is not a guarantee that dengue is eradicated and will soon be

eradicated because the source of infection, the dengue-carrying mosquitoes, Aedis

agypti, are still out there waiting for a chance to spread the dengue virus. In barangay

Calawisan, around 27 cases of dengue was recorded early this year and in Kalawisan

Elementary School, where the experiment is conducted, there were 5 schoolchildren

suffered the said infection that has alarmed the school and its community. This

1
alarming data have provoked us researchers to conduct a study regarding the larvicidal

potential and effectiveness of plant’s phytochemical or acid compounds against dengue-

carrying mosquitoes, Aedis aegypti, and we found that acid compounds in Oregano

plant.

Oregano (Origanum vulgare) a very common tropical plant that grows anywhere

in tropical countries such as the Philippines has been investigated for its larvicidal

efficacy which is being used in this study as a larvicide against Aedis egypti.

Many have been done to eradicate these tiny harmful vectors, one of which is

the use of insecticidal fumes which has proven to have dangerous effects to other

organisms and to the people who are living nearby the treated area. Humans, when

exposed to pesticides are prone to acquire serious ailments like cancer and even birth

defects. Aside from the hazardous effects it brings, it is also expensive that ordinary

Filipinos cannot just easily purchase even one for their family’s protection against these

little assassins. It is, therefore, very necessary to identify a safe, eco-friendly,

economical, accessible yet effective alternate source of larvicide in order to reduce

mosquito menace which is the main purpose of this study. If proven effective and

efficient, the urgent call to reduce mosquito imminence will be addressed.

2
 Review of Related Literature

Long before the discoveries of commercial larvicides to kill insects’ larvae, plants

have always been a constant partner of people in their everyday endeavours. According

to the World Health Organization (WHO) survey, 80% of the populations living in the

developing countries rely almost exclusively on traditional medicine for their primary

health care. Plants are considered as a rich source of bioactive chemicals and they may

be an alternative source of mosquito control agents (Rabha et al., 2007).

Phytochemicals derived from plant sources can act as larvicide, insect growth

regulators, repellent, and ovipositor attractant and have a different activities observed

by many researchers (Murugan, 1998). Plant alkaloids have been found to affect

physiological systems in higher animals as well as in insects (Saxena and Tikku 1990). A

large number of plant essential oils may be potential sources of mosquito larvicides

because they constitute a rich source of bioactive components (Lahlou et al. 2001, Cetin

et al. 2004). Natural products are the best option because of their less harmful nature

to environment and non-targeted organisms. Several extracts and compounds from

different plant families have been evaluated to show new and promising larvicides

(Markouk et al., 2000; Prabakar et al., 2004; Mohan and Ramaswamy, 2007; Innocent

et al., 2008a and 2008b), however very few plant products have been developed for

controlling mosquitoes. Given these facts, the researchers are provoked to study the

benefits we can get from plants in order to solve the existing dilemma our country and

3
barangay are facing nowadays and dengue is undeniably frightening and petrifying the

community since it has killed the infected residents ranges from infants and even

adults.

Dengue-carrying mosquito as popularly known by people has a scientific name

Aedis aegypti. According to the National Center for Emerging and Zoonotic Infectious

Diseases, Division of Vector-Borne Diseases, they described Aedis aegypti mosquitoes

as small, dark mosquito with white lyre shaped markings and banded legs. This is most

frequently found in tropical and subtropical areas of the world and is historically

considered to be a primary vector of viral diseases such as dengue fever, chikungunya,

Yellow Fever and Zika infection. Severe dengue (also known as Dengue Haemorrhagic

Fever) was first recognized in 1950s during dengue epidemics in the Philippines and

Thailand. Today, severe dengue affects most Asian and Latin American countries and

has become a leading cause of hospitalization and death among children and adults in

these regions according to the World Health Organization. WHO considered dengue as

a global burden since incidence has grown dramatically around the world with an

estimated 390 million dengue infections recorded per year. Another study, of the

prevalence of dengue, estimates that 3.9 billion people, in 128 countries, are at risk of

infection with dengue viruses (Brady OJ, Gething PW, Bhatt S, Messina JP, Brownstein

JS, Hoen AG et al., 201). Given this alarming data, the domination of these tiny vectors

which bring dengue must be stopped but the use of insecticides and other chemical

substances that can control them is very hazardous to human health and to the non-

4
target organisms. So, to answer the urgent call for an eco-friendly, cost-effective, safe

and economical source of larvicide, people always turn to plants.

One of the plants that can aid humans in controlling these vectors is the Oregano

(Origanum vulgare). This plant is also known as Wild Marjoram, Mountain Mint,

Origanum, Wintersweet and Winter Marjoram. This erectly spreading plant has strong

aromatic characteristics, with leaves and stems that are fleshy. The leaves of oregano

are heart-shaped, with toothed edges, and which, grow for up to 9 meters in length.

Oregano contains an acid compound Carvacrol and thymol which are investigated for

their larvicidal and insecticidal activity. Similarly, Carvacrol has been reported to be a

very potent larvicidal compound, more toxic than Thymol, when tested against An.

Stephensi (Lee et al., 1997). L. sidoides essential oil and its main constituent Thymol

were shown to be very active against A. aegypti larvae (Carvalho et al. 2003). These

thymol and carvacrol acid compounds or essential oils are found in Oregano (Origanum

vulgare). May this study would pave way to the discovery of the eco-friendly,

economical, yet cost-effective alternative source of larvicide against Aedis aegypti.

5
STATEMENT OF THE PROBLEM

This study aims to determine the larvicidal efficacy of Oregano (Origanum

vulgare) powdered dried leaves against Aedis aegypti. This experimental research

sought to answer the following queries:

1. What is the pre-test result before the treatment of the following groups:

a. Experimental Group (Origanum vulgare); and

b. Control Group (Antimos 1)?

2. What is the post-test result after the treatment of the following groups:

a. Experimental Group (Origanum vulgare); and

b. Control Group (Antimos 1)?

3. Is there a significant difference between the post-tests results of the same

groupings?

STATEMENT OF THE HYPOTHESIS

There is no significant difference between the post-tests results of the

experimental and the control group.

6
RESEARCH DESIGN

This is an experimental study employing the efficacy of Oregano (Origanum

vulgare) powdered dried leaves as a larvicide against Aedis eagypti mosquitoes. The

data were taken after the pre-test and post-test treatment given to the Aedis egypti

vectors in three replicates. In each replicate, there are two groups of Aedis egypti in

second larval instar where there is the experimental group (treated with the powdered

dried leaves of Oregano) and a positive control group (treated with a commercial

product, Antimos-1). This research investigation utilized quantitative and qualitative

approaches.

RESEARCH ENVIRONMENT

The realization of this research was conducted in two locations:

In Kalawisan Elementary School, situated at Zone Sunlot, Calawisan, Lapu-Lapu City,

where the specimen, second instar larvae of dengue-carrying mosquitoes (Aedis egypti)
were taken. A jar of clean water was placed beside in one of the classrooms at
Kalawisan Elementary School and waited for the mosquitoes to lay eggs. The
experiment was also conducted in the same site where the specimens were taken.

University of San Carlos, Talamban Campus, was the location for the specimen
identification. The mosquito larvae were sent there for proper identification in the
Biology Department. The powdered dried leaves of Oregano (Origanum vulgare) was
also identified by the Biology Department of the said university.

7
RESEARCH SPECIMEN

The sample specimens of the experimental study were randomly selected in a jar

of clean water placed beside in one of the classrooms at Kalawisan Elementary School,

where the researchers are studying and alarming cases of dengue have been reported

every year. There were many larvae in the jar, nevertheless, only 60 larvae were used

in the experiment. The experiment was in three replicates, 10 larvae were placed in

each plastic cup. The specimens must be second instar larvae of the dengue-carrying

mosquito (Aedis egypti).

RESEARCH INSTRUMENTS

Only the apparatus necessary for the specimen identification were acquired from

the Biology Department of the University of San Carlos, Talamban Campus. On the

other hand, some of the instruments used in the conduct of the study are readily

available in the school.

Two Medicine Droppers were used to transfer the specimen from its cultured habitat

in to the plastic cups for experimentation. These were used for the proper transfer of

the specimens and to avoid exerting force on the larvae which may cause its death

since second instar larvae are still very delicate.

8
6 Plastic Cups, where the specimens were placed for experimentation and treatment.

Plastic Jar with clean water, where mosquito larvae were cultured before

experimentation and treatment.

Two Magnifying Glasses were used to check if there are already larvae in the jar of

water.

Two Reagent Bottles, where the powdered dried leaves of the Oregano (Origanum

vulgare) were placed after pounding.

Three Mortars and Pestles were used in grinding and powdering the air-dried leaves

of Oregano (Origanum vulgare)

Two Plastic Trays were the Oregano (Origanum vulgare) fresh leaves were placed.

The leaves must be air-dried.

9
RESEARCH PROCEDURES

GATHERING OF DATA

Sampling Procedure

The sample specimens of the experimental study were randomly selected

in a jar of clean water which were identified as second larval instar of the dengue-

carrying mosquito, Aedis egypti. The Oregano (Origanum vulgare) leaves were obtained

in the school vicinity and in the Barangay, where the study is conducted. Oregano plant

is very common in the said Barangay that you can see it in anywhere within the

Barangay’s jurisdiction, in the nearby Barangays and even in the whole country and

neighboring countries since it is a tropical plant.

The researchers sought the expert services of the Biology Department of the

University of San Carlos, Talamban Campus, for the proper identification of the species

of the mosquito larvae and the Oregano (Origanum vulgare) leaves.

2. Preparation of the Powdered Dried Leaves

The Oregano (Origanum vulgare) fresh leaves were randomly gathered in the

vicinity of Barangay Calawisan where the plant is very common and widely spread. This

is where Kalawisan Elementary School situated, where the study is conducted. The

were placed in a tray and were air – dried. The air – dried leaves of Oregano (Origanum

10
vulgare) were manually grounded and powdered using mortar and pestle. After being

grounded, the powdered leaves were placed in a clean Reagent bottles for storage

before use.

3. Preparation of the Plastic Cups

The plastic cups were washed properly with distilled water. After washing, 200

mL of distilled water was poured in each plastic cup where second instar mosquito

larvae are to be placed for experimentation.

4. Transfer of the Aedis aegypti Larvae

Using the medicine droppers, the larvae were transferred from its cultured

habitat in to the plastic cups filled with 200 mL distilled water. There must be 10 second

instar larvae in each plastic cup.

5. Location of the Experimentation

The six plastic cups with ten second larval instar of the Aedis aegypti were

placed inside a classroom.

6. Before the Treatment (Pre-Test)

After transferring the Aedis aegypti second instar larvae from its cultured habitat

to the plastic cups, the Aedis aegypti second instar larvae were left for 24 hours without

treatment. After 24 hours, the larvae were observed and observations were recorded.

11
7. Application of the Powdered Dried Leaves of Oregano (Origanum vulgare) and

Commercial Larvicide, Antimos 1

Pour 3 grams of powdered dried leaves of Oregano (Origanum vulgare) into the

plastic cups marked with A which is the Experimental Group while in the plastic cups

marked with B, pour 3 grams of commercial larvicide, Antimos 1. This is done after the

24-hour pre-test period. The pouring of the powdered dried leaves of the Oregano

(Origanum vulgare) and commercial larvicide, Antimos 1, must be done at the same

time. After applying treatment to both groups, they are left for another 24 hours.

8. Chemical Content of the Oregano (Origanum vulgare)

Oregano (Origanum vulgare) leaves contains acid compounds, Carvacrola nd

Thymol, which have been investigated for its larvicidal property against mosquito

larvae.

9. Statistical Tool

To give an in-depth to the experimental study, the comparison of the pre-test

and post-test results were tabulated and calculated numerically based on the

effectiveness of the Oregano (Origanum vulgare) powdered dried leaves as a larvicide

against dengue-carrying mosquitoes (Aedis egypti). T-test was applied to test the

significant differences in the larvicidal effects of Oregano (Origanum vulgare) powdered

dried leaves as compared to the commercial product, Antimos 1.

12
 DEFINITION OF TERMS

To take a full grasp of this study, it would be best to know the terms used, they

are defined operationally:

Efficacy – or effectiveness; it is the ability to produce a desired or intended result.

Aedis aegypti – a yellow fever mosquito, is a mosquito that can spread dengue fever,

chikungunya, Zika fever, and yellow fever.

Larvicide – is an insecticide that is specifically targeted against the larval life stage of

an insect and is commonly used in mosquitoes.

Oregano – is an herb that has juicy stems and leaves, that has scientific name

Origanum vulgare. Its smell is similar to a lemon scent. Its leaves and stems

has tiny hairs making it feel like a rough texture. It is high in Thymol and

carvacrol which are a very strong antioxidant.

Dengue Fever – a debilitating viral disease of the tropics, transmitted by mosquitoes,

and causing sudden fever and acute pains in the joints that may lead to

death.

Larvae – the active immature form of an insect, especially one that differs greatly from

the adult and forms the stage between egg and pupa. In the case of

13
 mosquitoes, it undergoes 4 larval instars.

Pre-test – the test administered before the treatment.

Post-test – the test administered after the treatment.

Experimental Group – the group in which the powdered dried leaves of Sugar Apple

(Annona squamosa L.) is applied.

Positive-Control Group – the group treated with the commercial larvicide , Antimos1.

14
 PRESENTATION, ANALYSIS AND INTERPRETATION OF

DATA

Larvicidal Efficacy of Oregano (Origanum vulgare) Powdered

Dried Leaves against Aedis aegypti

STATEMENT OF THE PROBLEM

This study aims to determine the larvicidal efficacy Oregano (Origanum vulgare)

powdered dried leaves against dengue-carrying mosquitoes, Aedis aegypti. This

experimental research sought to answer the following queries:

1. What is the pre-test result before the treatment of the following groups:

a. Experimental Group (Origanum vulgare); and

b. Control Group (Antimos 1)?

2. What is the post-test result after the treatment of the following groups:

a. Experimental Group (Origanum vulgare); and

b. Control Group (Antimos 1)?

3. Is there a significant difference between the post-tests results of the same

groupings?

15
STATEMENT OF THE HYPOTHESIS

There is no significant difference between the post-tests results of the

experimental and the control group.

Results: Accept – No Significant Difference

RESULTS AND DISCUSSION

Table 1

The Pre – test result of the number of larvae in each plastic cup before the treatment.

Treatment Subjects Number % of % of Remarks

R
Or of Larvae Dead Live
Group Larvae Larvae
1 A 10 0 100% Negative
Experimental 2 A 10 0 100% Negative
3 A 10 0 100% Negative
1 B 10 0 100% Negative
Control 2 B 10 0 100% Negative
3 B 10 0 100% Negative

The data above indicate the initial number of Aedis aegypti larvae in its second

larval instar placed in a plastic cup with distilled water. There are 10 Aedis aegypti

larvae in each plastic cup for both Experimental Group and Control Group. All set-ups

received a negative remark or there is no change occurred since the subjects were not

yet exposed to the two treatments.

16
 Table 2

The Post – test result of the number of larvae in each plastic cup after the treatment.

There are ten second instar larvae in each cup.

Treatment Subjects Number of % of Remarks

Or R Dead Dead
Group Larvae Larvae

1 A 7 70% Potent
Experimental 2 A 8 80% Potent
(Origanum 3 A 7 70% Potent
vulgare)
1 B 9 90% Effective
Control 2 B 8 80% Effective
(Antimos 1) 3 B 8 80% Effective

Table 2 reveals the post-test results of the number of Aedis aegypti in

second larval instar after the treatment. It indicates that the Oregano (Origanum
vulgare) powdered dried leaves kills an average of 73% of the second instar larvae
while the Antimos 1, which is a commercial larvicide shows a remarkable performance
that sweeps away an average of 83% of the larvae. Based on the table, Oregano
(Origanum vulgare) powdered dried leaves is a potential larvicide against Aedis aegypti
larvae. Kishore et al (2011) reviewed the efficacy of phytochemicals against mosquito
larvae according to their chemical nature and described the mosquito larvicidal
potentiality of several plant derived secondary materials, such as, alkanes, alkenes,
alkynes and simple aromatics, lactones, essential oils and fatty acids, terpenes,
alkaloids, steroids, isoflavonoids, pterocarpans and lignans.

17
 Table 3

Post-tests difference between the Experimental and the Control Group

No. of Sq. Diff

Treatment/ Diff T- P-
R N Death (X-M)² Decision Interpretation
Group (X) (X-M) Value Value

1 10 7 -0.33 0.11

A. Experimental 2 10 8 0.67 0.44

(Origanum
vulgare)
Accept Not
3 10 7 -0.33 0.11 -2.12 0.05
Ho Significant
M: 7.33 SS: 0.67

1 10 9 0.67 0.44
B. Control 2 10 8 -0.33 0.11
(Antimos 1) 3 10 8 -0.33 0.11
M: 8.33 SS: 0.67

Table 3 reveals the difference in the larvicidal efficacy between the Oregano

(Origanum vulgare) powdered dried leaves and the commercial larvicide, Antimos 1.

The computed t-value is -2.12 which is lower than the p-value at 0.05 level of

significance which is 0.05. The hypothesis which states that there is no significant

difference between the post-tests results of the experimental and the control group

against Aedis aegypti second larval instar is accepted.

18
 The result implies that the Oregano (Origanum vulgare) powdered dried leaves is

not as effective as the commercial larvicide, Antimos 1, but can be the best alternative

source of larvicide against Aedis aegypti mosquito species since it also showed a

remarkable performance after killing 73% of the second instar larvae of the Aedis

aegypti in the Experimental group. Thymol and carvacrol are acid compunds present in

Oregano (Origanum vulgare) plant. Therefore, Oregano plant, if air-dried and

powdered, may serve as the best alternative source of larvicide against dengue-carrying

mosquitoes, Aedis aegypti.

CONCLUSION

There is no significant difference between the larvicidal efficacies of the Oregano

(Origanum vulgare) powdered dried leaves and the commercial larvicide which is

Antimos 1 against Aedis aegypti second larval instar. The potency of the powdered

dried leaves of the Oregano (Origanum vulgare) is not as high as the commercial

larvicide, Antimos 1. However, results show that the powdered dried leaves of the

Oregano (Origanum vulgare) could be used as an alternative source of larvicide against

Aedis aegypti larvae.

19
RECOMMENDATION

The experimental study necessitates the following recommendations:

1. Information generated from the results should be disseminated.

2. Plants found in the locality with known larvicidal property/potency should be

collected and used in the study for comparative purposes.

3. Further experimental study on the powdered dried leaves of the Oregano (Origanum

vulgare) with different concentrations is highly suggested to determine what

concentration is the most effective and also for comparative purposes.

4. Another experimental research about the powdered dried leaves of the Oregano

(Origanum vulgare) to another mosquito species is also suggested to prove its

effectiveness.

5. Further comprehensive investigation and tests of the Oregano (Origanum vulgare)

powdered dried leaves is suggested to produce a novel commercial larvicide using

Oregano leaves.

6. Further experimental study on the extracted dried leaves of Oregano (Origanum

vulgare) using ethanol and methanol is suggested to prove its effectiveness.

20
 ACKNOWLEDGEMENT

The success and final outcome of this project required a lot of guidance and

assistance from many people and we are extremely privileged to have got this all along

the completion of our Research Study or Science Investigatory Project. All that we have

done is only due to such supervision and assistance and we would not forget to thank

them.

We respect and thank Mrs. Susan R. Obiedo, the school principal of Kalawisan

Elementary School for providing us an opportunity to do the research study in the

school and for giving us all the support and guidance which made us complete the

research study duly. We are extremely thankful to her for providing such a nice support

and guidance, although she had busy schedule managing the school.

We owe our deep gratitude to our research study adviser, Mrs. Noveim T.

Leoligao, who took keen interest on our study and guided us all along, till the

completion of our study by providing all the necessary information we need in the

whole duration of the project, from data mining or researching, collecting the specimen,

conducting the experiment, down to interpreting the data result.

We are thankful to and fortunate enough to get constant encouragement,

support and guidance from our parents and family members which motivated and
helped us in successfully completing our research study. Above all, we would like to
praise, glorify and thanks our Creator, Lord Jesus, for giving us the wisdom we needed

and good people who served as our support system in conducting the research study.

21
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24
 Republic of the Philippines
Region VII, Central Visayas
Department of Education
Division of Lapu-Lapu City
District 8

KALAWISAN ELEMENTARY SCHOOL

Larvicidal Efficacy of Oregano

(Origanum vulgare)
Powdered Dried Leaves
against Aedis aegypti
Researchers
Bona Faith S. Bonganciso
Karlo B. Quinain
Princess Jona T. Lastimosa

Research Adviser
Noveim T. Leoligao
 Appendix A
COMPUTATION

Treatment 1

N1: 3
df1 = N - 1 = 3 - 1 = 2
M1: 7.33
SS1: 0.67
s²1 = SS1/(N - 1) = 0.67/(3-1) = 0.33

Treatment 2

N2: 3
df2 = N - 1 = 3 - 1 = 2
M2: 8.33
SS2: 0.67
s²2 = SS2/(N - 1) = 0.67/(3-1) = 0.33

T-value Calculation

s2p = ((df1/(df1 + df2)) * s21) + ((df2/(df2 + df2)) * s22) = ((2/4) * 0.33) + ((2/4) *
0.33) = 0.33
s2M1 = s2p/N1 = 0.33/3 = 0.11
s2M2 = s2p/N2 = 0.33/3 = 0.11

t = (M1 - M2)/√(s2M1 + s2M2) = -1/√0.22 = -2.12

25
Appendix 2

PHOTOS

26
The pouring of 200 mL water into the plastic cups
 The transferring of
the larvae from the jar of water into the plastic cups
 TABLE OF CONTENTS

Title Page Pages

Chapter 1: The Problem and its Setting

Rationale

The Problem

Statement of the Problem

Statement of the Hypothesis

Definition of Terms

Chapter 2: Review of Related Literature

Chapter 3: Research Methodology

Research Design

Research Environment

Research Respondents

Research Instruments

Data Gathering Procedures

Data Analysis

Acknowledgement

References

Appendices