1

Sampaguita ​(Jasminum sambac) ​Extracts with Calamansi ​(Citrofortunella microcarpa)​ Extracts

as an Effective Alternative to Citronella ​(Cymbogon nardus) ​Repellent Against Mosquitoes

Fairy Alyjah C. Dumilon

Denise Anne D. Estacio

Kelsey Loren B. Menil

Mariel C. Tamaray

De La Salle University Integrated School

23 March 2020

 
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Abstract

The present study was conducted to determine the mosquito repellent activities of four different

naturally-occurring substances and find a locally-sourced and more affordable alternative for the

commonly used repellent, citronella. This study aims to produce a powerful repellent by

associating the extracts of two endemically known products in the Philippines: sampaguita

(​jasminum sambac) ​and calamansi ​(citrofortunella microcarpa)​. A human testing procedure

consisting of numerous setups that will last for 3 minutes each was performed to obtain credible

results. The effectiveness of each repellent, both individually and combined, was determined by

observing and recording the number of mosquitoes that lingered on the human arm for more than

10 seconds. In conclusion, recorded results had exhibited that the combined extracts of

sampaguita and calamansi has proven to be more effective as compared to citronella.

Furthermore, observations have shown that the sampaguita extracts alone was the most effective

repellent among the rest due to its effects on the mosquitoes while the citronella extract was the

least effective repellent. In spite of the successfully answered research questions, certain

limitations and mishaps regarding the effectiveness of the sugar-fermenting yeast, the handling

of the mosquitoes, and the number of mosquitoes used were also experienced throughout the

process.

Keywords​: repellent, mosquito, natural, endemic

 
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Chapter 1: Introduction and Literature Review

Introduction

The Philippines is currently battling a nationwide mosquito-borne epidemic, the dengue

virus. Citizens and researchers alike are finding ways to repel and eliminate the ​Aedes aegypti

mosquito, the largest carrier of dengue. With limited resources and the growing resistance of

mosquitoes to many man-made repellents, the need for accessible, cheap, and natural alternatives

continues to increase rapidly. Natural repellents made of locally-sourced components are ideal to

support the income and livelihood of local farmers as well as the economy of the Philippines.

Citronella microcarpa​ (calamansi) comes from the family of citrus, which are known to be

natural mosquito repellents because of their strong scent. ​Jasminum sambac​ (sampaguita) is a

Philippine flower known to have a mild, sweet scent which can be used to balance out the strong

tones of citrus. Both plants are endemic in the Philippines and their extracts have shown

larvicidal and insecticidal activities that may aid the repelling of mosquitoes. Their availability to

the public may guarantee its lower cost as compared to manufactured man-made repellents. This

study seeks to combine sampaguita plant and calamansi extracts to produce a strong repellant

that may rival the potency of repellants made of only ​Cymbopogon nardus ​(citronella).

Statement of the Problem

This research paper aims to answer the following questions:

1. Is the mixture of sampaguita and calamansi an effective mosquito repellent?

 
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2. Will calamansi and sampaguita individually serve as an effective mosquito

repellent?

3. At what length of time and percentage will each setup be effective in repelling

mosquitoes?

4. Which setup is most effective in repelling mosquitoes?

Significance of the Study

The study on the potency of sampaguita and calamansi extract to repel mosquitoes can be

an effective means to reduce the chances of obtaining dengue and any mosquito-related diseases.

The objective of the study is to produce a more effective, cheaper, and accessible alternative

mosquito repellent for the masses; to expand the uses of sampaguita and calamansi being a

natural product with health-benefiting properties; and utilizing the prevalent and

locally-available native products of the Philippines. The experimental repellant aims to prevent

people of all ages, especially dengue victims, from mosquito bites and possibilities of obtaining

mosquito-related diseases. This grants the middle-class and the marginalized community to a

more accessible product since sampaguita and calamansi are endemic and cost-efficient products

present in the Philippines. This also benefits the economic activity in the Philippines and the

local farmers of San Pedro to make use of the indigenous nature, repelling properties, and other

beneficial attributes of sampaguita and calamansi.

 
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Literature Review

The Effectivity Span of the Different Concentration Levels of the Phytochemical Extracts

from Peels of Citrus Fruits Against Insects ​(Kelsey Menil)

The article “Mosquito Repellent Activity of Phytochemical Extracts from Peels of Citrus

Fruit Species” (2012) states that mosquitos have been proven to be driven away by the

phytochemical extracts contained inside the fruit peels of citrus fruits but only at a reasonable

concentration. The mosquito repellent activity of phytochemical extracts from the fruit peels of

five different citrus fruit species, Citrus sinensis, Citrus limonum, Citrus aurantifolia, Citrus

reticulata, and Citrus vitis, was tested. The exhibition of repellent activity of the phytochemical

extracts by application on some bare portions of the body (legs, face, and hands) of the human

volunteers produced a positive outcome. All the extracts from the different species of citrus fruits

resulted to an effectivity of repellent activity in their individual concentrations though with

unequal levels of time duration. According to the acquired data, the 5% and 10% concentration

of all the extracts failed to produce any repellent effect while the 25% concentration of the Citrus

vitis extracts produced a long-lasting repellent effect of more than 5 hours. As for the 15% and

20% concentration levels of all the extracts, these exhibited a repellent effect of less than 1 hour

and more than 2 hours respectively. Side effects such as mild skin itching and sneezing due to

the use of the extracts from the peels of Citrus sinensis, Citrus limonum, Citrus reticulata, and

Citrus aurantifolia were observed after the experiment. In spite of the minimal mishaps and

reactions detected, this study has proven that phytochemical extracts from citrus fruits guarantees

repellency against insects generally.

 
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This article will be the basis on why calamansi can be considered as an alternative for

citronella mosquito repellents due to its citric nature or the citrus components it contains. The

various concentration levels tested will be the footing on the appropriate amount of

phytochemical extracts of calamansi that are to be used in the mixture in order to determine the

effectivity span and repellency.

The Larvicidal and Insecticidal Activities of Citrus Microcarpa and Jasminum Sambac on

the Mortality Rate of Dengue Virus Vector Aedes Aegypti (​Mariel Tamaray)

Students from the Lyceum of the Philippines University in Batangas conducted a research

study on natural insecticides using four Philippine-native plants tested on the leading dengue

carrier in the Philippines, the mosquito Aedes aegypti. This was done to determine which among

the four was the most effective. Two of those plants are the​ ​Citrus Microcarpa (calamansi) and

the Jasminum Sambac (sampaguita), both proven in previous studies to have shown larvicidal

activities and contain elements which are also used in potent commercial agricultural pesticides.

From their findings, calamansi extract was the most effective, having eradicated 80% of the

mosquito’s larvae within 24 hours and have shown its effectiveness within two hours of exposure

to the larvae. This was only achieved, however, using a very high concentration of the extract, at

500ppm concentration. Among the four plants, calamansi was the only extract to have shown

significant levels of very high larvicidal activity, showing results that may deem calamansi as a

potent alternative to commercial insecticides. It is relatively cheap, given that it is

locally-produced and it is safe to humans and the environment as opposed to man-made

insecticides. Sampaguita, another plant tested against the larvae, have caused less than 9%

mortality within 24 hours of exposure with the same 500ppm concentration, as found by the

 
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student researchers. They concluded that sampaguita has no potential activity for control of

larvae regeneration and that there exists very little larvicidal activity within sampaguita extract

that can effectively destroy mosquito larvae. All the four plants were tested against black pepper,

the positive control, which performed more efficiently than the rest, having eliminated 100% of

the larvae within 24 hours. However, black pepper extract contains toxic components that may

be harmful to non-target organisms. Considering the advantages and disadvantages of each plant,

those who wish to use or produce mosquito repellants can be well-aware of the effects of various

repellents to humans, the environment, and other organisms.

Calamansi as a Mosquito Repellent ​(Fairy Dumilon)

According to the research done by Lee Najiah (2009), calamansi prohibits 7 bacterial

strains such as Escherichia coli, Citrobacter freundii, Aeromonas hydrophila, Pseudomonas

aeruginosa, Streptococcus agalatiae, E. Tarda, and Yersinia enterocolitica. Thus, calamansi

might have potential as a microbial agent for aquaculture use. The presence of major compounds,

limonene had contributed immensely to the aroma, antibacterial, antifungal and insect repellent

properties. The quality of Citrus essential oil depends highly on the composition of limonene

present in the essential oil which may be found in different types of citrus fruits. The effect of

hydrodistillation extraction process on the yield and limonene composition of essential oil

extracted from peels of calamansi lime had been studied and the potential application of Citrus

essential oil as natural aromatic gel was examined in this research. The Citrus essential oil had

been extracted from various Citrus peels using hydrodistillation method for 3, 4, 5, 6 and 7

hours. The Results obtained showed that the extraction time will affect the yield and limonene

composition in Citrus essential oil significantly (p < 0.05) in the hydrodistillation process.

 
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Sensory evaluation had been carried out to evaluate the natural aromatic gel made. Based on the

results, the criteria of preciseness, intensity and persistence had been rated above moderate level

while rating for overall acceptability was above the level of slightly like. Hence, Citrus essential

oil has the potential in making natural aromatic gel which can become a substitute for synthetic

air freshener in daily life.

The Use of Textiles in the Process of Korean Food and Drug Administration Guidelines for

Assessing Mosquito Repellents ​(Denise Anne Estacio)

The article “Comparison of Repellency Effect of Mosquito Repellents for DEET,

Citronella, and Fennel Oil” (2015) elaborates on the effectivity of the procedures of KFDA done

to test the repellency of DEET, citronella, and fennel oil to mosquitoes. The KFDA guidelines

were based and improved from the guidelines of the Environmental Protection Agency,

American Society Testing and Materials, and the World Health Organization Pesticide

Evaluation Scheme, and was approved by the ​institutional review board​ of Konkuk University

Hospital. The study compares the benefits, time length of protection, and percent protection of

the chemical repellent, DEET; and the botanical mosquito repellents, citronella oil and fennel oil,

extracted from Eucalyptus maculata citriodora and Foeniculum vulgare. In order to test the

repellency of the solutions, the procedure involved monitoring mosquito-human contact in a test

cage where one of the arms is applied with the repellent and the other is not. The untreated arm

was first tested. For 3 minutes, it was placed in the test cage and the number of mosquito

landings and bitings were recorded. As for the DEET-treated arm, it was inserted in the test cage

for 3 minutes for 1 hour interval and lasted up to 6 hours, while each of the botanical

repellent-treated arms was placed in the test cage for 3 minutes for 1 hour intervals that lasted for

 
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2 hours. Mosquito landings and bitings were recorded for each set up, the untreated arm’s

exposure for 3 minutes and the treated arms’ for every hour. The repellency was assessed by

multiplying a 100% to the difference of the number of the bites in the DEET-treated arm

deducted by the number of bites on the botanical based repellent over the number of bites on the

DEET-treated arm. The complete protection time, on the other hand, was observed from a

different set up. Each treated-arm was placed in the test cage for 3 minutes for every 10-minute

interval until the first mosquito came in contact after the treatment. Although a written consent

was initially given before the experiment to inform the volunteers of the threats, the process was

concerning because it involves the risk of putting the patients in harm when bitten by a

disease-carrying mosquito.

An article entitled “Methods of Imparting Mosquito Repellent Agents and the

Assessing Mosquito Repellency on Textile” (2016)​ ​discusses the variety of fabrics that are

effective to be treated with mosquito repellant without damaging its quality under different

conditions; the potency of mosquito repellent even when not directly applied to human skin; and

the chances of minimizing unwanted side effects when repellents, especially chemical-based, are

applied to the skin. The study claims that fabrics can be used as a physical-barrier for mosquitoes

to prevent them from having direct contact with humans while still being able to test the

repellency of the product. The standard fabrics that were used and showed positive results in the

study were cotton, polyester and blended fabrics (denim). These fabrics were treated with the

solution by four different methods namely: absorption technique where the repellent is sprayed

or pad dried to the fabric or the fabric is dipped into the solution, the repincorporation where the

fabric is initially treated with the repellent during the manufacturing process, polymer coating

 
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and microencapsulation which are similarly characterized with the textile being coated with

layers of polymers. Pad drying and microencapsulation were argued to be more effective. The

study suggests that the use of fabrics when testing the repellency of mosquito-fighting products

is beneficial and guarantees safety with the appropriate and qualified type fabrics applied with

the suitable repellent agent with the proper methods.

These articles will be the basis for our methodology in testing mosquito repellents. The

guidelines of Korean Food and Drug Administration will be used for the step-by-step procedures,

and the use of fabric as a physical-barrier for human skin contact to mosquitoes will ensure the

safety of our subjects while still being able to effectively test the repellency of the solutions.

 
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Chapter 2: Methodology

The purpose of this study is to identify a botanical plant that will serve as a more potent

mosquito repellent to Citronella and that is more prevalent and available to the Philippines. A

comprehensive experimental setup was done in a laboratory in De La Salle University Integrated

School to four tested and qualified subjects. The procedure for this methodology is based on the

Korean Food and Drugs Administration modified from the WHOPES and EPA methods, but

slight alterations were made according to the available time, resources, and equipment. The

repellents used for this experiment were citronella, calamansi, sampaguita, and a mixture of

sampaguita and calamansi. Qualified subjects were gathered to record and observe the number of

mosquitoes and the timespan before a mosquito makes contact with the arm. The data gathered

were used to test the repellency percent and the complete protection of the solutions. To identify

the repellency percent, the formula based on KFDA guidelines was modified according to the

available time and resources. The repellency was assessed by multiplying a 100% to the

difference of 1 by the number of bites on the botanical based repellent over the number of bites

on the control arm. They were then ranked correspondingly, tested using the Chi-Square

Goodness-Of-Fit test, and presented in a bar graph and table with respect to each research

question.

 
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Experimental Setup

 
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Chapter 3: Findings and Discussion

Research Question #1: Is the mixture of sampaguita and calamansi an effective mosquito

repellent?

The objective of our study is to formulate a natural mosquito repellent that would rival

citronella​—​a research-supported, widely-marketed natural mosquito repellent​—​in terms of

affordability, effectiveness, and accessibility. The group decided to make citronella the standard

for effective mosquito repellents since it has been commercially sold and proven to be effective.

Sampaguita oil, calamansi extract, and a mixture of the two were tested against citronella.

Table 1

​The number of mosquitoes that touched the human arm within 3 minutes and the timespan

before a mosquito makes contact with the arm (complete protection time)

No. of Mosquitoes that touched

Repellent Complete Protection Time (CPT) 
within 3 mins 

Citronella  4 20 s

Sampaguita-Calamansi  0 55 s

Calamansi  4 44s

Sampaguita  2 > 10 mins

 
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4
C itronella Repellency(%) = (1 − 10
) × 100% = 60%

0
S ampaguita − C alamans Repellency(%) = (1 − 10
) × 100% = 100%

Based on Table 1, the sampaguita-calamansi mixture has the highest repellency rate

while sampaguita has the best protection time. It is worthy to note that the results of citronella

under repellency rate is inaccurate because several mosquitoes have been crushed during its

setup, implying that the repellency rate may be smaller than claimed. Notice that the complete

protection time of the sampaguita-calamansi mixture is significantly longer than citronella’s,

further supporting the antithesis that the mixture may be a more effective repellent than the

citronella.

Research Question #2: Will calamansi and sampaguita individually serve as an effective

mosquito repellent?

Based on the results listed on Table 1, we find that the repellency rate and CPT of

calamansi and sampaguita are as follows:

2
S ampaguita Repellency(%) = (1 − 10
) × 100% = 80%

4
C alamansi Repellency(%) = (1 − 10
) × 100% = 60%

Both the calamansi and sampaguita proved to be effective mosquito repellents, easily surpassing

the potency of citronella. Notably, sampaguita exceeded the expectations of the researchers.

Sampaguita turned out to be the most effective repellent. This may be due to the sampaguita

being an oil substance, thus holding condensed amounts of fragrance that easily repels the

 
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mosquitos. On the other hand, the Calamansi extract had also shown promising results of

repellency against mosquitoes in comparison to Citronella in terms of complete protection time.

This was due to its acidity and citrus scent.

As for the Sampaguita-Calamansi solution, pure calamansi as a thick liquid may have

diluted the sampaguita oil in the mixture, but its fragrance remained intact, thus making it more

effective in comparison to the Calamansi solution.

Research Question #3: At what length of time and percentage will each setup be effective in

repelling mosquitoes?

Given the formula and recorded time in Table 1 the repellency percent of the solutions

are as follows: Citronella at 60% in 20 seconds, Calamansi at 60% in 44 seconds, Sampaguita at

80% in more than 10 minutes, and Sampaguita-Calamansi at 100% in 55 seconds. Presumably, if

the procedures were executed at 1-hour intervals for 4 hours, it is safe to hypothesize​ t​ hat the

repellency percent will decline as the time progresses.

N o. of bites in the treatment arm

Repellency(%) = 1 − N o. of bites in the control arm
× 100%

4
C itronella Repellency(%) = (1 − 10
) × 100% = 60%

4
C alamansi Repellency(%) = (1 − 10
) × 100% = 60%

2
S ampaguita Repellency(%) = (1 − 10
) × 100% = 80%

0
S ampaguita − C alamansi Repellency(%) = (1 − 10
) × 100% = 100%

 
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Research Question #4: Which setup is most effective in repelling mosquitoes?

According to recorded data on Table 1, the Sampaguita was the most potent setup, with

its complete protection time lasting more than 10 minutes as compared to the other three that

only lasted for a few seconds. In addition to the repellency percent and complete protection time,

the group had observed that whenever a mosquito attempted to make contact with the hand when

the Sampaguita setup took place, the mosquito dropped down and failed to fly afterwards.

Furthermore, twitching of legs and irritation were observed. The mosquitoes became weak and

had temporarily lost consciousness. A few minutes after the subject’s hand was removed, the

mosquitoes regained their consciousness and were able to move normally again. In the midst of

the complete protection time process, it took more than ten minutes for a mosquito to endure the

Sampaguita’s powerful scent. In conclusion, only two mosquitoes were able to successfully

linger on the subject’s hand during the Sampaguita setup, making it the most effective repellent

among the other setups.

Figure 2

Complete Protection Time of Each Repellent

 
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Figure 3

Number of Mosquitoes that Made Contact with the Subject’s Arm within the First 3 minutes after

being Situated with the Repellents

Figure 4

Repellency Percent of Each Extract

 
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Chapter 4: Conclusion and Recommendations

Conclusion

This study was able to test four chosen substances for mosquito repellency and identify

the most effective among the four. It was observed that mosquitoes were completely repelled

from the arm by the Citronella for 20 seconds, Calamansi for 44 seconds, Sampaguita-Calamansi

mixture for 55 seconds, and Sampaguita for more than 10 minutes. Using the repellency

percentages, the Chi-square Goodness-Of-Fit test showed that there is no significant difference

between the number of mosquitoes that touched the arm within the first 3 minutes among the 4

substances. It disproved the hypothesis that citronella would be the most effective, considering

the number of studies that highlight its function as a repellent. This study was also able to

produce surprising results—that sampaguita, an overlooked substance in many mosquito

repellent testing, has proven to be the most effective of the four. Data of this research also shows

that Sampaguita and Calamansi are individually more potent than Citronella as a mosquito

repellent; therefore, Sampaguita plant induced with Calamansi extracts is indeed a much more

effective mosquito repellent in comparison to Citronella.

Recommendations

There were several notable errors done by the researchers during the experiment: First,

the repellents were obtained in different ways. The citronella solution and sampaguita solution

were obtained at a market and were commercially marketed as mosquito repellent and fragrance

oil respectively. On the other hand, the calamansi solution was manually extracted from the fruit

 
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itself. Their differences in consistency and possible chemical add-ons may have had affected

their effectiveness as a mosquito repellent. It is recommended that all solutions must be extracted

using proper extraction methods and equipment. Second, several mosquitoes expired in the

process of extracting the container from the 6-litre bottle. This was due to the limited space in the

6-litre bottle and obscure movement of the mosquitoes. To avoid crushing the mosquitoes, the

researchers may acquire a bigger​ s​ torage and more mosquitoes. Third, given the insufficient time

due to the limited life span of the mosquitoes, the experiment was done in less than 3 hours, but

the initial duration of the experiment is said to be done for more than 5 hours. The experiment on

the repellency percent must be done after 1 hour of intervals. Ideally, the set up must also be

repeated to obtain accurate results and observe for errors. To avoid affecting the actual results of

the experiment, researchers may conduct the experiment ahead of time with a complete set of all

required materials. Fourth, the recorded experimental complete protection time for all setups

were comparably lower than the expected complete protection time based on research. Fifth, the

group did not consider the effects that the solution may cause when directly situated in the

subjects’ arm considering their skin conditions. Sixth, given the limited time, resources, and

equipment, the experimental setup done by the group was comparably different from the setup

done in the research. They are different in size of the container and number of mosquitoes. These

errors have made minimal-to-moderate effects on the overall results of the experiment.

Due to the limitations of the scope of this research, future studies could be done on the

effects of high amounts of citronella and calamansi to human skin, ways to dilute the acidity of

the calamansi to avoid itchiness when directly situated on human skin without decreasing its

 
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repellency to mosquitoes, improving the study on the effectiveness of the sugar-fermenting yeast

based on mosquitoes’ feeding time when sugar is their only option for feeding.

 
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References

Anuar, A., & Yusof, N. (2016, June 28). ​Methods of imparting mosquito repellent agents and the

assessing mosquito repellency on textile.​ Fashion and Textiles, 3(1), 1-14. Retrieved from

https://fashionandtextiles.springeropen.com/articles/10.1186/s40691-016-0064-y

De Villa, L. C., Abantes, M. A., Asi, M. C., Balmeo, N. C., Bustillo, A. D., Calangi, E. M., &

Cruzado, L. R. (2012). ​Larvicidal activity of four Philippine plants against Dengue virus

vector Aedes aegypti(Linn.)​. Retrieved from

https://research.lpubatangas.edu.ph/wp-content/uploads/2014/10/STETH-6.2.pdf

Effiom, O. E., Avoaja, D. A., & Ohaeri, C. C. (2012). ​Mosquito repellent activity of

phytochemical extracts from peels of citrus fruit species.​ Global Journal of Science

Frontier Research, 12(1), 1-8. Retrieved from

https://globaljournals.org/GJSFR_Volume12/2-Mosquito-Repellent-Activity-of-Phytoche

mical-Extracts.pdf

Liew, P. Y. (2014). ​Study on the effect of hydrodistillation on yield and limonene composition of

various citrus essential oil and its application as natural aromatic gel. Pahang: UMP.

Retrieved from

https://pdfs.semanticscholar.org/16f3/7c1e9a06e629830ca7350b653f7d4a8b0d86.pdf?_g

a=2.36016617.897944693.1567995795-685577998.1567995795

Smallegange, R.C., Schmied, W.H., van Roey, K.J. et al (2010, October 25). ​Sugar-fermenting
yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles
gambiae.​ Malar J 9, 292. Retrieved from doi:10.1186/1475-2875-9-292

 
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World Health Organization. (2009). ​Guidelines for efficacy testing of mosquito repellents for

human skin​ (No. WHO/HTM/NTD/WHOPES/2009.4). Geneva: World Health

Organization. Retrieved from

https://apps.who.int/iris/bitstream/handle/10665/70072/WHO_HTM_NTD_WHOPES_20

09.4_eng.pdf?sequence=1&isAllowed=y

Yoon, J. K., Kim, K. C., Cho, Y., Gwon, Y. D., Cho, H. S., Heo, Y., ... & Kim, Y. B. (2015).

Comparison of repellency effect of mosquito repellents for DEET, citronella, and fennel

oil.​ Journal of parasitology research. Retrieved from

https://pdfs.semanticscholar.org/0dcd/e6ded44dd2a9dc26bdba6e264b7426e3d445.pdf

 
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Appendix A: Procedure

Materials:

1. Five boxes of 20 female ​Aedes aegypti​ mosquitoes.

2. 5 clear tupperwares with the lids cut off and covered with 5 thin mesh, and a small hole

bore onto the side

3. 5 cotton balls soaked in sugar-water or honey diluted in water

4. Five separate sprayer bottles containing 20ml of each individual extracts (Citronella,

Calamansi, and Sampaguita) and 10ml of each extract for the Sampaguita-Calamansi

solution

5. Five stockings attached to the cut top area of five 6-Litre bottles using elastic bands.

6. Odorless Soap

7. Clean Cloth

8. Stopwatch

9. Timer

10. Mosquito Racquet Killer

11. Elastic bands

12. Mesh

Procedure:
1. Acquire 5 boxes of female mosquitoes, each box containing 20 mosquitoes, in a lab
facility near Los ​Baños​.
2. Prepare the 5 tupperwares. Bore a large hole in the tupperware’s lid and cover it with
mesh. Bore another small hole into the side of the tupperware then insert the cotton ball
after dipping it in sugar-water or honey diluted in water.

 
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3. For 3 days, the mosquitoes are placed in their respective tupperware; isolated and
incapable of feeding blood. The sweetened cotton ball will serve as their nourishment,
hence should be replaced everyday.
4. Acquire the botanical repellents. The repellents used for the experiment are citronella,
calamansi, sampaguita, and a mixture of sampaguita and calamansi.
5. Prepare the 6-liter bottles and the stockings. Cut the top most area of the 6-liter bottles,
then attach the stockings in the cut area and secure with elastic bands. Put individual
tupperwares containing the mosquitoes for each 6-litre bottle.
6. Prepare the repellents by putting 20 mL of each into a spray bottle. As for the
calamansi-sampaguita solution, put 10 mL of each substance then mix well.
7. Free the mosquitoes in the passage, then immediately extract the tupperware out of the
bottle and secure the passage by tying it to a knot. Make sure that all mosquitoes from the
container were released before taking the container out and also avoided killing the
mosquitoes in the process.
8. Collect qualified representatives. Instruct them to rinse their hands and arms using
odorless soaps, then dry with a clean cloth. Without any repellent, cover the
representative’s arm with the cut hose. Insert the covered arm into the insertion.
9. For 3 minutes, observe and record the number of mosquitoes that lingered on the arm for
more than 10 seconds. Remove the subject’s arm. To identify if the representative is
qualified for the experiment, they must have their arms landed and rest for 10 seconds by
at least 10 mosquitoes to ensure that the mosquitoes are stimulated by their scents.
10. Repeat the process with the tested subjects, but with the repellents. Spray 10 mL of the
solution on the subject’s arm before covering with the cut hose.
11. Apply the same process for the complete protection time. Use the stopwatch, to record
the time when the first mosquito lands and stays for more than 10 seconds on the
subject’s arm.

 
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12. After the experiment, in the event that the mosquitoes did not die from the solution,

exterminate the mosquitoes with the use of a mosquito racket killer or the most effective

solution.

 
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Appendix B: Experiment Log