Research Proposal Paper

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

SYNERGISTIC LARVICIDAL EFFECT OF Origanum vulgare L. (OREGANO) AND Cymbopogon citratus (DC.) Stapf (LEMONGRASS) OIL EXTRACT ON Culex MOSQUITO LARVAE

A Research Study Presented to the Senior High School Department

De La Salle University Dasmariñas City of Dasmariñas, Cavite

In Partial Fulfilment of the Requirements for the Senior High Research Badajos, Gabrielle Mae A. Calalang, Kyle B. Caparangca, Adrian Miguel B. Enriquez, Allisha Jana C. Pasuquin, Stephanie Fiona P. Tolog, Louise Nicole R. October 2022

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

THE PROBLEM AND ITS BACKGROUND CHAPTER 1

This chapter includes a brief introduction regarding the research, the problem that it aims to solve, and the variables that will be used to determine the synergistic effect of the Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf oil extract on Culex mosquito larvae.

Introduction

Mosquitoes are small flying insects that belong to the order of insects known as Diptera or true flies. They are widely known to spread vectorborne diseases, which have killed more people than all wars combined, making them the most dangerous animal on earth. According to the WHO (2022), a parasitic infection transmitted by Anopheline mosquitoes called malaria and dengue fever, which is transmitted by Aedes mosquitoes, were the most prevalent diseases that were caused by mosquitoes that resulted in more than 400,000 fatalities annually. Furthermore, othermosquito-borne diseases, such as filariasis, chikungunya fever, Zika virus fever, yellow fever, West Nile fever, and Japanese encephalitis, have resulted in the deaths of tens of millions of people.

The Culex family of mosquitoes is known as one of the most common types of mosquito species which are vectors of several known deadly diseases The Culex mosquito species are best known for the spread of

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Saint Louis, the Japanese variant of encephalitis, and West Nile virus which has affected over 40,000 people in the United States since 1999 (Aranda, 2017). These types of mosquitoes are commonly found in households and prefer to feed on humans and animals, preferably birds, at night (BCM, 2017; County News Center, 2016). Additionally, Culex mosquitoes typically live in places with temperate and tropical climates, such as the Philippines. These species prefer to breed in highly polluted habitats like sewage and drainage systems, containers such as waste tires and rain barrels, and stagnant surface water habitats (Valent BioSciences, [n.d.]).

According to the Centers for Disease Control and Prevention (2022), a female Culex mosquito can produce hundreds of eggs laid in the shape of a raft. Within 7 10 days, these eggs transition into adult mosquitoes that could possibly carry deadly diseases. The larva is second to the four life stages of a mosquito. It is the stage where mosquitoes can be easily exterminated as it is the most vulnerable phase of their life. Furthermore, mosquito larvae shed their skin about four times in their larval stage. According to the American Mosquito Control Association [n.d.], the stages between the shedding of their skin are known as instars. The larval stage of a mosquito has four instars, and when the fourth instar larva sheds its exoskeleton, it transitions into a pupa.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Insecticides are developed to kill one or more species of insects, which cause disruption to a mosquito’s nervous system, damage its exoskeletons, and control them in ways that prevent it from engaging in destructive behaviors (National Pesticide Information Center, 2019). Furthermore, larvicides, a type of insecticide used to control mosquitoes, also came to light. These chemicals may come in liquid products or solids, which include tablets, pellets, and granules. According to the CDC (2020), larvicides work by killing mosquito larvae even before it matures into fully grown mosquitoes. Currently, no single method is enough to lessen the global burden of disease caused by mosquitoes. As it may cause nutritional deficiencies and harmful effects on one’s endocrinal, reproductive, respiratory, and neurological systems, synthetic pesticides are now thought to be a considerably riskier and more dangerous substitute for plant based bioinsecticides (Yadav and Devi, 2017). Therefore, it is imperative that new, safer, and more efficient methods of controlling mosquitoes be created.

With the presence of harmful effects of commercially available insecticides and larvicides, plant-based larvicides are great alternatives. Specifically, the researchers would like to focus on the synergistic effect of the Cymbopogon citratus (DC.) Stapf (lemongrass) and Origanum vulgare L. (oregano). Both plants were proven to be insect killers by multiple researchers. Reddy et al. (2018) conducted a study that tested the larvicidal

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

effect of Cymbopogon citratus (DC.) Stapf on Aedes mosquito larvae and found out that it did affect the larvae. Furthermore, the results were also consistent with the study of Araonu et al. (2018), which tested the plant on the Anopheles gambiae mosquito. Like Cymbopogon citratus, (DC.) Stapf, research such as the study of Govindarajan et al. (2016), which tested the carvacrol and terpinen 4 ol of the Origanum vulgare L. against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus. In their research, they stated that they might be considered eco friendly larvicides. This study was further supported by the research of Bouguerra et al. (2019), as it revealed that the abundance of carvacrol in the dried part of the plant (oregano) is the main component that exerts toxicity to adult mosquitoes and eggs.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Theoretical/Conceptual Framework

Figure 1. Research paradigm of the study.

This study utilizes the use of the Input Process Output (IPO) Method.

As seen in Figure 1, Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. are the two main plants that will be used for experimentation. Additionally, Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. will be compared to Oregano only and Lemongrass solely oil extracts with regards to their effect on the mortality rate of Culex mosquito larvae and time of effectivity. The process of oil extraction through hydro distillation will be used to measure the total mortality rate of Culex mosquito larvae, the

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

essential oils extracted from the plants’ time of effectivity, and determine if there is a significant difference between Oregano oil extract, Lemongrass oil extract, and the combined extracted oils of Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. Additionally, the experiments and observations will all be conducted at the De La Salle UniversityDasmariñas laboratory. The expected output and findings from this study is the synergistic larvicidal effect of Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. in Culex mosquito larvae.

Statement of the Problem

Mosquitoes belong to the family of Culicidae and are known to be insects that fly. The blood-sucking habits of these insects make them the deadliest animal in the world, spreading diseases such as malaria and dengue. (CDC, 2019). To prevent the spread of these types of diseases, larvicides are introduced and utilized to target mosquito larvae before they mature into blood sucking adults. (CDC, 2020). Chemically made insecticides have adverse biological effects on the environment highlighting the need for healthy and ecologically friendly insecticides (Abutaha et al., 2018). Studies by Mariam et al. (2021) and Bouguerra et al. (2019), established that Cymbopogon citratus (DC.) Stapf (lemongrass) and Origanum vulgare L. (oregano) have acted as ingredients of larvicides.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

This research aims to determine the synergistic larvicidal effects of Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. in Culex mosquito larvae. Specifically, this study aims to answer the following questions:

1. What is the time of effectivity of the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract on Culex mosquito using 25 mg/L and 50 mg/L concentration?

2. What is the total mortality rate of the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract on Culex mosquito using 25 mg/L and 50 mg/L concentration?

3. Is there a significant difference between the effectiveness in terms of time of effectivity and total mortality rate of the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract to the following:

a. Oregano oil extract

b. Lemongrass oil extract

Hypotheses of the Study

Null Hypothesis H0; There is no significant difference between the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract to an oregano only oil extract in terms of their time of effectivity.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Null Hypothesis H0; There is no significant difference between the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract to a lemongrass only oil extract in terms of their time of effectivity.

Null Hypothesis H0; There is no significant difference between the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract to an oregano only oil extract in terms of their total mortality rate.

Null Hypothesis H0; There is no significant difference between the combined Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. oil extract to a lemongrass only oil extract in terms of their total mortality rate.

Significance of the Study

This study will contribute to further research in fighting against mosquitoes. Moreover, it will also benefit the following:

Family households. As mosquitoes can grow anywhere, this study will help free family households from mosquitoes with the use of organic larvicides that have less harmful effects on humans and the environment.

Communities. As the presence of mosquitoes is not limited to an individual’s house, this study will be beneficial to the community for it will bring awareness to the utilization of safer mosquito pesticides, particularly larvicides.

Entrepreneurs. Through this study, businesses could use the combination of Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

oil extract for a new product that can kill mosquito larvae at their immature stage with less health and environmental risks.

Future Researchers. The information that the study offers may be a useful reference for future researchers that would want to pursue topics the study is aligned with. Moreover, the results that this study gathered may serve as a springboard for future and further research.

Scope and Delimitations

The study will utilize experiments and observations as the method of data gathering among 120 Culex mosquito larvae which will be conducted at the De La Salle University - Dasmariñas laboratory in a span of one (1) day. The Culex mosquito larvae will be collected from the University of the Philippines Las Baños (UPLB).

The study will only focus on the synergistic larvicidal effect of Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf oil extract on the female 4th instar Culex mosquito larvae. Specifically, the time of effectivity and mortality rate of the mentioned plant oil extracts with 25 mg/L and 50 mg/Lconcentrations on the female 4th instar Culex mosquito larvae. This study will not cover other types and the whole population of mosquitoes, environmental effects of the formulated organic larvicides, and the effects of other external factors such as temperature and food on the Culex mosquito larvae. However, to avoid the death of the Culex mosquito

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

larvae due to starvation, it will be fed biscuit dried yeast with a ratio of 75:25 by weight.

This study does not aim to determine and identify the chemical composition of essential oils extracted from the plant samples. Thus, this study will not utilize Gas chromatography-mass spectrometry analysis, an instrumental method that uses gas chromatograph (GC) coupled to a mass spectrometer (MS) to separate, identify, and quantify complex chemical mixtures.

Definition of Terms

For a better understanding of the study at hand, the following terms are defined in the context of the study:

Culex mosquito larvae. The organism which the researchers aim to test the larvicides.

Combined Essential Oil. The combination of the essential oil of the Cymbopogon citratus (DC.) Stapf and Origanum vulgare L.

Cymbopogon citratus (DC.) Stapf. The scientific name of lemongrass and one of the plants that will be used in the study.

Clevenger Apparatus. The device that will be used in essential oil extraction.

Hydro distillation. The method that will be used to extract essential oils from the plant samples.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Larvicidal effect. The similarity of the time of effectivity and total mortality rate of the combined essential oil to a simple oregano and lemongrass oil extract.

Origanum vulgare L. The scientific name of oregano and one of the plants that will be used in the study.

Simple Oil Extract. The separate oil extract of the oregano and lemongrass essential oil; each plant oil extract is treated independently.

Synergistic Effect. The results that will be obtained when the essential oils of Cymbopogon citratus (DC.) Staph and Origanum vulgare L. are combined.

Time of effectivity. The average of the duration from the application of the larvicide to the death of the larvae; this data will be observed every two (2) minutes for an hour.

Total mortality rate. The amount of mosquito larvae that will die from the larvicide after an hour which will be determined by dividing the total number of mosquito larvae exposed to a particular extract's concentration by the number of dead larvae, multiplied by 100%.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

CHAPTER 2

REVIEW OF RELATED LITERATURE

The related literature from journals, scholarly articles, textbooks, and electronic journals and books are presented and discussed throughout this chapter. This is categorized into subtopics and concludes with a synthesis.

Mosquito Prevalence in the Philippines and the World

Mosquitoes are insects belonging to the order Diptera and the suborder Nematocera. These are little, two-winged insects that can reach a size of 15 mm. The mosquito genera Anopheles, Culex, Aedes, Ochlerotatus (previously a subgenus of Aedes), and Mansonia, which belong to the subfamilies Anophelinae and Culicidae (with approximately 2000 species), are prominently concerned with disease transmission and are known as a scourge that affects both humans and animals.

Mosquitoes are notorious for being a nuisance and for transmitting vector-borne diseases, which continue to be the leading cause of illness and death (Alayo et al., 2015). Mosquito-borne diseases such as malaria, dengue fever, chikungunya fever, and others have killed more people than all wars combined. As a result, the WHO listed mosquitoes as the world's number one public enemy and the most hazardous animal on earth. Moreover, mosquito borne diseases are prevalent in over 100 nations worldwide, afflicting over 700,000,000 people each year, including

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

40,000,000 Indians. According to WHO Africa, the Region continues to bear a disproportionately large percentage of the global malaria prevalence. In 2020, Africa had 95% cases of malaria and a 96% mortality rate. Around 80% of all malaria deaths in the Region were caused by children under the age of five. Nigeria (31.9%), the Democratic Republic of the Congo (13.2%), the United Republic of Tanzania (4.1%), and Mozambique (3.8%) accounted for slightly more than half of all malaria deaths globally.

Because the Philippines is a rich, tropical, and biodiverse location, numerous mosquitoes and small insects are swarming around. Several mosquito-borne diseases, notably dengue fever, malaria, filariasis, and chikungunya affect the Filipinos. The Philippines has recorded high morbidity and mortality rates because of Dengue fever. The Department of Health (DOH) announced a national dengue outbreak in 2019, reporting 146,062 cases registered from January to July 2019. Western Visayas, CALABARZON, and Central Visayas were the most highly affected provinces (International Federation of Red Cross and Red Crescent Societies, 2019). Chikungunya is another mosquito-borne disease that the DOH is monitoring. Currently, there is no known treatment for the disease, and all drugs are aimed at alleviating symptoms (WHO, 2017). During the first half of 2017, most cases were discovered in the provinces of Romblon, Aurora, Zambales, Batangas, and Laguna. The mosquito Aedes species

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

also carries the Zika virus, which causes a similar illness to influenza and is accompanied by conjunctivitis (WHO, 2018).

Culex Mosquito Prevalence

The Culex mosquito, generally known as common house mosquitoes, is the most prevalent mosquito species worldwide (Bhattacharya et al., 2016). It frequently takes blood meals from animals, including people, and are key carriers of new and re emerging pathogens (Barba et al., 2019). Moreover, the Culex mosquitoes are a diverse group of mosquitoes that are commonly found in temperate climate zones and are known vectors of several arboviruses, including West Nile virus (WNV), St. Louis encephalitis virus (SLEV), Avian malaria, Rift valley fever, Lymphatic filariasis, eastern equine encephalitis virus, and western equine encephalitis virus (Linthicum et al., 2016; Bellone and Anna Bella, 2020).

Culex mosquitoes, specifically Culex quinquefasciatus, are very common in tropical and subtropical metropolitan areas where environmental circumstances support their proliferation. Vork and Connelly (2016) described the Culex larvae to have a wide head and an oval gill that can be found at the bottom of its long antennae with large grass-like tips. Wherein Souza (2017) added that, although Culex and Aedes larvae are almost identical, the body of Culex’ is “hairy” and its siphon is longer and considerably lighter in color than Aedes’

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

In India and Cambodia, WNV carried by Culex quinquefasciatus mosquitoes have been reported (Paramasivan et al., 2003; Auerswald et al., 2020), whereas SLEV in the presence of Culex quinquefasciatus mosquitoes have been confirmed in Mexico, Argentina, and Brazil (Luby, 2017). Japanese Encephalitis Virus is primarily found in India, China, and Southeast Asia. Although there have been no reports of significant virus transmission by these mosquitoes in the Philippines, it is nevertheless critical to keep an eye out for and reduce their populations since these mosquitoes are potential vectors of Japanese B Encephalitis. Moreover, Wuchereria bancrofti, a filarial worm, has been detected throughout the country in Culex quinquefasciatus.

Culex Mosquito Larvae Behavior, Habitat, and Growth

The life of a Culex mosquito starts when an adult female lays its eggs on water. According to the CDC (2022), the eggs float on water and form a raft by sticking together 100 300 eggs. After it hatches, it reaches the next stage of its life cycle, the “larva,” which are also called “wigglers.” CDC described the mosquito larvae to be very active. They asserted that during this stage, these larvae also shed their skin multiple times. For food, CDC stated that it lives off of a variety of things found in the water. Additionally, El Husseiny et al. (2018) observed in their study that it can showcase cannibalism if it were under starvation.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

For their habitat, Nanjul et al. (2018) concluded that the best environment for these larvae to grow in is dirty water. This was also supported by Wilkerson et al. (2021), who stated that Culex pipiens live in highly eutrophic waters. Moreover, Amusan and Ogbogu (2019) stated that there are more mosquito larvae during dry seasons than wet seasons, which was consistent with Omolade and Adetutu’s (2018) study. Furthermore, there are also other factors that can influence the population and community of Culex mosquito larvae, such as the larval density, temperature fluctuations, solute concentrations, salinity and pH level (Alcalay et al., 2018; Ruiz-Santiago et al., 2018).

Larvicidal Effects of Other Plant Species on Mosquitoes

The use of larvicide, a type of insecticide, is one of the methods for controlling the population of mosquitoes. It is through killing the mosquito larvae before it reaches adulthood (CDC, 2020). Plant based larvicides are a great alternative to pesticides available on the market, as the chemical components of synthetic pesticides can cause harmful effects on living organisms and the environment. According to O'Malley G.F. and O'Malley R. (2022), numerous insecticides can cause poisoning if swallowed, inhaled, or absorbed through the skin. Coughing, breathing difficulties, eye tearing, and even heart problems are some of the symptoms that can be observed when exposed to these chemicals. Aside from that, the wide use

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

of certain synthetic insecticides has caused insecticide resistance in some mosquitoes, including Aedes mosquito species, which are vectors of the dengue, yellow fever, chikungunya, and Zika virus (Smith et al., 2016).

The use of plant-based insecticide is a good alternative and has many advantages. According to Souto et al. (2021), it is eco-friendly, biodegradable, and less toxic to humans. Ocimum basilicum, also known as basil, is a herbaceous and aromatic plant from the Lamiaceae family and is primarily found in Asia and Africa (NParks, 2022). A study by Rudayni et al. (2021) found that O. basilicum can be a potential larvicide to control the population of mosquitoes. The study tested the effectiveness of the ethanolic extract of Basil (O. basilicum) leaves and flowers against the larvae of Anopheles arabiensis and Culex quinquefasciatus mosquitoes. Rudayni et al. found that the flower extract of O. basilicum has more potency and biological activity than the basil leaves extract and is more toxic to the larvae of An. arabiensis compared to C. quinquefasciatus. The researchers have also observed the damage that was caused by the extracts on the mosquito larvae. They were able to point out that the O. basilicum extracts have caused discoloration and disconnected and swollen alimentary canal of the larvae. In addition, a study made by Chan et al. (2022) has shown that the hexane extract of O. basilicum leaf can be an effective larvicide against the third instar larva of Aedes albopictus. The larvae were exposed to six different concentrations from 6.25 to 200 µg/mL in a 24 and 48 hour

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

exposure period. During the 24 hour exposure period of the Ae. albopictus larvae from the 200 µg/mL of Hexane extract of O. basilicum leaves, the researchers have observed that the mosquito larvae experienced blackening of the abdomen and extended cephalo-thoracic junction. Chan et al. (2022) have concluded that the hexane extract of the O. basilicum leaves has shown significant larvicidal activity against Ae. albopictus larvae.

Furthermore, Mentha x piperita, commonly known as peppermint, is a flowering plant from the Lamiaceae family that also has the potential to regulate the mosquito population (Bellassoued et al., 2018). A study by Iqbal et al. (2022) found that the Mentha piperita L. leaves extract could be a potential substitute for synthetic larvicides against Culex quinquefasciatus larvae. The researchers tested the effectiveness of the ethanolic and aqueous extract of M. piperita L. leaves against all instar larvae of C. quinquefasciatus mosquito. Iqbal et al. found that both extracts showed high larvicidal activity. The researchers have also observed that as the concentration of the M. piperita L. extracts grew, the number of dead larvae increased. Moreover, the study by El-Kasem Bosly (2022) tested the efficiency of the essential oils of three plants from the Lamiaceae family, including M. piperita L, as potential larvicides against the third instar larvae of Culex pipiens. The researcher determined that among the three essential oils, peppermint (M. piperita L.) ranked second, with a 92% mortality rate during the 24 hour exposure period.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

In addition, de Oliveira et al. (2021) conducted a study about the larvicidal activity of Origanum Vulgare L. and Thymus vulgaris L. against the Aedes aegypti L. In their study, they concluded that the two essential oils were lethal. The O LC50 values in Ae. Aegypti were reported to be 37.5 g/mL. Egyptian larvae T and 38.9 g/mL for vulgare, respectively. vulgaris. Adult mosquitoes had LC50 values of 14.3 and 11.7 g/mL, respectively. O. offensive essential oils from Elegans and T. Results for Vulgaris ranged from 8.9% to 37.8% and 4.4% to 68.9%, respectively, demonstrating a high repellency effect. Under an optical microscope, the morphological variations between the control group and the EO-treated larvae were also discernible. The results suggest that O. T from Sacramento and unpleasant essential oils. Vulgaris may be a viable source for the development of fresh insecticides for the control of Ae. Aegypti.

Cymbopogon citratus (DC.) Stapf, Origanum vulgare L., and their components

Cymbopogon is a genus with about 55 species of grasses prevalent in temperate and tropical climates, which includes Cymbopogon citratus, commonly known as lemongrass. It is an herbaceous perennial grass that is commonly used as a culinary and medicinal herb that is abundantly found in Sri Lanka and India. In addition, people extract essential oils from their leaves which contain insect repellency. Particularly, the leaves and

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

branches of lemongrass contain the essential oil called citral (3,7 dimethyl 2,6 octadienal). Citral is an E isomer diastereoisomer (geranial or citral A) with its Z isomer known as the neral or citral B which can be extracted with the use of hydro distillation. According to a study by Almusaed A. and AlSamaree S. in 2017, the extracted lemongrass essential oil or lemonal has the formula C10H16O which belongs to monoterpenoids. After distillation, citral oil can be used to produce ionones, vitamin A, and other citral acetals, which have several uses in perfumery and can aid in antibacterial reduction.

In a study by Brügger et al. (2019), thirteen compounds or 95.98% of the overall composition of the lemongrass essential oil were identified. Neral (31.5%), citral (26.1%), nonan 4 ol (6.54%), camphene (5.19%), 6 metil hept 5 en 2 one (4.36%), citronelal (3.83%), caryophyllene (3.26%), citronelol (2.95%), caryophyllene oxide (2.63%), γ muurolene (2.46%), limonene (2.32%), geranyl acetate (2.27%), and geranial (2.15%) are the primary components of lemongrass oil. However, variations in the number of components observed, with geranial as its major compound, is dependent on the extracted organ, age, geographic location and extraction processes performed on the plant. Consequently, it was found in a study by Mukarram et al. (2021) that lemongrass essential oil (LEO) includes significant amounts of citronellal, citronellol, elemol, isogeranial, isoneral, geraniol, geranyl acetate, and germacrene-D, all of which possess

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

medicinal benefits which include antifungal, antibacterial, antiviral, anticancer, and antioxidant properties.

Origanum vulgare is a perennial herb that grows up to 80 cm tall and is widely known as oregano with dark oval, aromatic leaves, and spikes of white, pink, or purple flowers. It is also called marjoram from the family of Lamiaceae which originated in the Mediterranean with a long history of utilization as food and medicinal plants. The study by Kumar et al. in 2016 established that β-bisabolene, carvacrol, caryophyllene, limonene, linalool, p-cymene, c-terpinene, ocimene, terpinene, and 4-terpineol are all found in O. vulgare leaves essential oil. Furthermore, O. vulgare leaves extract contains carvacrol, flavonoids, rosmarinic acid, phenol carvacrol, tannins, thymol, and triterpenes as bioactive constituents. It also contains calcium, copper, iron, magnesium, niacin, thiamine, and vitamins. One of the primary essential oil molecules produced by oregano plants is carvacrol [2-methyl5-(1-methylethyl) phenol], a monoterpenic phenol, which has powerful antimicrobial action against foodborne pathogens. Like Cymbopogon citratus, oregano oil possesses antioxidant, antibacterial, antifungal, antiparasitic, and antimicrobial qualities. This was also supported by a study by the Association for the Advancement of Restorative Medicine (AARM), which claims that Origanum vulgare is beneficial to human health as it possesses antioxidant and antibacterial activities in both in vitro and in vivo

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

models. Additionally, Origanum vulgare oil has been utilized in the food and cosmetic sectors to effectively prevent growth and reproduction in order to improve and prolong product shelf life.

Larvicidal Effect of Cymbopogon citratus (DC.) Stapf and Origanum vulgare L. in Culex Mosquitoes and Other Mosquito Species

Cymbopogon citratus, otherwise known as lemongrass, is a plant that contains phytochemicals that can act as a larvicide. As stated by Mariam et al. (2021), this plant is known for its harmful effects on mosquito larvae. C. citratus contains Tannin and Saponin, which are phytochemicals that are toxic to mosquito larvae (Waris et al., 2020).

A study conducted by Muthukumar et al. (2021) showed the larvicidal effect of C. citratus (lemongrass) against Culex mosquito species. The study tested methanol and water extract of lemongrass at 50, 100, 150, and 200 mg/L concentrations in the third and fourth instar stages of the development of Culex mosquito larvae. The larval mortality rate was continuously recorded after 24, 48, and 72 hour exposure periods and was calculated using Abbott's formula (introduced in 1925). The result of the study showed that the Cymbopogon citratus extracts have a notable effect on the mortality rate of the larval stages of Culex mosquitoes. The study has also shown that the percentage of mortality in the third and fourth instar

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

larvae of the culex mosquito was higher in methanol extract than in the water extract of C. citratus. During the 24 hour exposure period, the third instar larvae have the maximum percentage of mortality of 84.60% at 200 mg/L, while the fourth instar larvae have the maximum mortality of 89.30% and 92.20% at 150 and 200 mg/L. Both results were observed in the methanol extract of C. citratus. During the 48 and 72 hour exposure periods, the third instar larvae have a maximum percentage of mortality of 90.20% and 94.20% at all tested concentrations observed in the methanol extract.

In addition, a study by Goselle et al. (2017) exhibited the larvicidal activity of Cymbopogon citratus from different methods of extraction, namely Maceration, Infusion, and Decoction against Culex mosquito larvae. The larval mortality rate was recorded at 2, 6, 24, and 48 hour periods in different concentrations of dried lemongrass (Maceration), wet lemongrass (Infusion), and boiled lemongrass (Decoction) extracts. The outcome has shown that the ethanol extract of dried lemongrass has the highest larval mortality at 100%. On the other hand, boiled lemongrass extract has larval mortality of 88.9%, followed by wet lemongrass extract at 62.2%.

Apart from the studies that have shown the effect of Cymbopogon citratus (lemongrass) on the larval stage of Culex mosquito species, the study of Mariam et al. (2021) has proved that the C. citratus has an effect on the larvae of Aedes mosquito species. The study is about the methanol

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

extract of lemongrass leaves and stalks against the fourth instar of Aedes aegypti larvae. After the extract treatment, at 2, 4, and 6 hour periods, 100% mortality at 7.5% extract concentration was observed, followed by 95% at 5% concentration and 55% at 2.5% concentration. The preceding result showed that C. citratus could be an effective larvicide against the larvae of the Aedes mosquito. Moreover, the study conducted by Seye et al. (2021) on the histopathological effects of C. citratus essential oil against the third instar larva of the Aedes aegypti mosquito has shown how lemongrass acts as a potent larvicide. According to Seye et al. (2021), the result of their study has shown that the essential oil of C. citratus damages and destroys the intestinal microvilli, muscle, and fat body mass.

Another study has also exhibited the effect of C. citratus on the larval stage of the Anopheles gambiae mosquito. The research by Njouku Tony et al. (2015) has observed that the root and leaf extract of the C. citratus was more toxic to the first instar larvae of An. gambiae than Culex quinquefasciatus and Aedes aegypti. The researchers stated that the An. gambiae mosquito grows well in freshwater compared to the other species that prefer polluted water. It indicates that the An. gambiae is less resistant to the extracts used. Furthermore, the study conducted by Batti et al. (2019) was about the different extracts of C. citratus and C. giganteus against the third and fourth instar stages of the development of An. gambiae larvae.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

The study has found that among the hexane, acetone, and methanol extracts of C. citratus and C. giganteus, the hexane extract of the two Cymbopogon species were the most harmful than the other extract.

On the other hand, de Oliveira et al. (2021) conducted a study about Origanum vulgare L. as an effective larvicide. In this investigation, the essential oils from the aerial portions of Origanum vulgare L. were employed. In addition to Thymus vulgaris L. were tested for their ability to repel, larvicide, and adulticide Aedes aegypti L. GC-MS analysis is used. The fundamental elements of the O The fundamental elements of the T Terpinen 4 ol (17.4%), carvacrol (16.0%), and thymol (10.4%) were the components of vulgare essential oil. The primary ingredients of the vulgaris essential oil were thymol (40.0%), p cymene (19.3%), and terpinene (17.3%). Both essential oils were lethal to Ae. reported O LC50 values in Ae were 37.5 g/mL. larvae of aegypti. T and 38.9 g/mL for vulgare, respectively. vulgaris. Adult mosquitoes had LC50 values of 14.3 and 11.7 g/mL, respectively. O. essential oils of elegans are offensive, and T. Results for vulgaris ranged from 8.9% to 37.8% and 4.4% to 68.9%, respectively, showing a strong repellency effect. Additionally, optical microscopy showed morphological differences between the control group and the EO treated larvae. The result suggests that the O. Vulgare and T. Vulgaris essential

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

oils can serve as a potential source for creating insecticides that will control the population of Ae. aegypti.

Extraction of Essential Oil from Cymbopogon citratus (DC.) Stapf and Origanum vulgare L.

Essential oils, also known as volatile odoriferous oil, are commonly extracted from different parts of one or more types of plants, which include their flowers, leaves, and stems. They are concentrated solutions made up of diverse mixtures of volatile chemicals and are used in various commercial products, including fragrances, pharmaceuticals, and pesticides. Before direct consumption of medicinal plants as herbal or traditional medicines, these plants must undergo the process of extraction. The method of extracting essential oils, or essential oil extraction, is characterized as attempting to extract certain substances from plants. Furthermore, medicinal plants go through the concept of preparation for experimentation by the proper and timely collection of the specific plant to be used. Aside from this, expert authentication is also necessary, as well as sufficient drying and grinding. Extraction, fractionation, and the isolation of the bioactive compound follows (Abubakar et al., 2020).

A study by Elyemni et al. (2019) on the “Extraction of Essentials Oils of Rosmarinus oficinalis by Two Different Methods: Hydro distillation and Microwave Assisted Hydro distillation” claims that the method of essential

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

oil extraction is generally obtained by performing two main techniques of extraction: azeotropic distillation (hydro distillation, hydro diffusion, and steam distillation) and solvent extraction. The study mainly focused on studying two methods of essential oil extraction which are: Clevenger hydro distillation (CH) and microwave-assisted hydro distillation (MAH). According to the same study, plant samples were collected when they were at the flowering stage wherein only the aerial part of the plant was used while letting its leaves and apical parts dry in the shade for a length of eight (8) days and a temperature of 25 degrees Celsius (25º C). The first method, Microwave-Assisted Hydro distillation (MAH), was performed employing a setup that involves a domestic microwave (MWD 119 WH, Whirlpool, China, 20L, 1100 W), a Clevenger-type extractor, and a cooling system to continuously condense the distillate. In order to add water back to the plant material, the additional condensed water was refluxed into the extraction flask. The method was carried out under favorable conditions such as time of extraction, microwave power, and the ratio of water to the plant material. The samples were heated within the microwave oven cavity while also being kept in a flask filled with distilled water that accelerates the process of extraction. On the other hand, the mixture was heated until every essential oil was extracted at constant power. The second method, Hydro distillation by Clevenger, also operated under optimum conditions. A quantity of the plant was added to a certain amount of distilled water in a flask and placed

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

in a balloon heater attached to a refrigerator. This action was taken to guarantee the occurrence of condensation of essential oils within a certain period. At the end, aqueous phase (aromatic water) and organic phase (essential oil) were observed. This method was verified with the study by Desai & Parikh (2015) as a promising alternative to traditional extraction methods due to how effectively it heats material up, how quickly energy is transferred, and how ecologically beneficial it is.

Similarly, the study of Wagh et al., (2021) focused on four (4) methods that can extract essential oils from lemongrass. The first two methods were the same as what the previous study used the Solvent Extraction Method and the Hydro distillation Method. The Solvent Extraction Method started when a dried lemongrass sample was put in a flask with a certain type of solvent (n hexane, a hydrocarbon solvent) and was left to stand for 36 hours to dissolve the essential oils. As the essential oils were extracted, they were poured into a container in the place of a beaker as ethanol was added to extract all the essential oil from the lemongrass. After, the mixture will be transferred to a separate container to allow the substances to reach equilibrium. The extracts were separated into two layers so that the ethanol extract could be collected and then discarded, leaving only the pure natural essential oils. A study by Majewska et al., (2019) pointed out that this method is quite efficient and relatively simpler. However, it requires a high volume of solvent that leads to unsatisfactory

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

reproducibility. An experimental study by Suryawanshi et al. (2016) and Alhassan et al. (2018) verified the effectiveness of Solvent Extraction as a method to extract essential oil from lemongrass leaves. Sometimes, the Soxhlet apparatus is utilized to extract lemongrass essential oil (LEO) using the Solvent Extraction Method (Alhassan et al., 2018). Soxhlet extraction involves continuous contact between the plant material and the refluxing liquid phase. This results in increased extraction efficiency. However, this method requires a long heating period at a high temperature usually near the solvent’s boiling point which may cause volatile chemicals to degrade thermally. With this, appropriate solvent selection is necessary to achieve a satisfactory extraction yield and to reduce the loss of volatiles.

Another essential oil extraction method is Hydro distillation which is carried out by adding fresh lemongrass samples and distilled water to a flask with a rubber stopper that was attached to the condenser and heating. Then, water is heated and is allowed to flow counter currently through the condenser up until it reaches the appropriate temperature. As the essential oil is eventually extracted and mixed with water vapor, the water vapor will be condensed and separated. The condensate was collected using a beaker and the mixture was separated by a separatory funnel. The method ended by collecting the oil and storing it in a bottle with a stopper. Most temperature sensitive substances that are insoluble in water and may disintegrate at their boiling point are processed using the steam distillation

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

method, which is the third essential oil extraction method in accordance with the same study. Additionally, it is currently the most famous method for extracting lemongrass essential oils and other plant oils.

The Steam Distillation Method includes putting the lemongrass sample and distilled water in a flask to be connected to a condenser and heat. Subsequently, water condensation shall occur that will be countercurrent to ensure steam. As the mixture reaches a temperature that will cause it to boil the extracted essential oil. Ice cubes are also used in this procedure to facilitate chilling and volatilization. This method is still a prominent preparative essential oil extraction of lemongrass. Lastly, the use of mortar and pestle to mash dried samples of lemongrass was performed to expose the inner stem of the plant. This technique is known as the Enfleurage Method where the smashed lemongrass is mixed with light flavored olive oil. Then, ethanol was added to the mixture which stood for 18 hours at room temperature for it to absorb the essential oil, leaving the olive oil and lemongrass residue behind. To remove the ethanol, the sample was kept in the water bath at 75-80 degrees Celsius (75-80º C) to determine the yield of oil.

Essential oil extraction of Origanum vulgare L. was able to utilize the same extraction method as lemongrass such as hydro distillation, steam distillation, Clevenger distillation, and microwave extraction. In the study of Bouguerra et al. (2019) about the extraction of Oregano Essential Oil, the

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

method of hydro distillation was utilized. 100 g of the plant's dried leaves were split up into small pieces. It was then hydro distilled in a Clevenger type of apparatus for 3 hours as per recommendation in the method by British Pharmacopoeia in 1988. The extracted essential oil was dried with the use of anhydrous sodium sulfate, and was kept in vials at 4°C. The total oils produced were calculated based on the dried weight of the plant materials.

A study by Hrnčič et al. (2020) used a variety of techniques which includes Ultra Sound Assisted Extraction. This method of extraction accelerates the secretion of essential oils from the plant as they allow for easier penetration of the solvents in the plant material. Through the same study, it has been observed that dry oregano can yield extracts rich in phenolic compounds with antioxidant activity when subjected to ultrasound assisted supercritical fluid extraction. The extracts' flavonoid profile remained unchanged, despite a rise in their overall phenolic content and an increase in their antioxidant power. As supercritical fluid extraction yields might be low, the use of ultrasound-assisted extraction was considered to be an effective method to enhance the mass transfer of mechanisms and kinetics.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Related Studies

Oregano Essential Oil as Potential Mosquito Larvicides

Mosquitoes are known to be responsible for the spread of diseases such as dengue, malaria, and yellow fever. Due to the continuous growth of its population, different processes of mosquito control and prevention came to light. Synthetic chemical insecticides have been released to the public market to achieve a mosquito free environment. Although insecticides perform their job of controlling pests, these chemicals put the environment in an unfavorable position and cause resistance to insecticides. To avoid all the problems brought about by these chemicals, scientists become more focused on safer alternatives made with plants.

Oregano is widely recognized as the major component of pesticides, particularly of mosquito repellents. A study by Bouguerra et al. (2019) utilized the essential oil of Algerian Origanum vulgare to determine its chemical composition and larvicidal efficacy against fourth instar larvae of Culex pipiens. The investigation began by gathering the necessary materials, including mosquitoes and plant materials. The C. pipiens mosquito larvae were collected from the laboratory of Applied Animal Biology and were reared. 20 samples of the larvae were stored in a pyrex storage jar with 150 ml of tap water, which was changed every three days and kept at a temperature between 25°C and 27°C The C. pipiens were fed with fresh food, a mixture of Biscuit dried yeast (72:25 by weight) daily.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

During the months of March to May, the researchers collected samples of the fresh aerial parts of the plant, O. vulgare. The research utilized the method of hydro distillation. 100 g of the plant's dried leaves were split up into small pieces. It was then hydro-distilled in a Clevenger type of apparatus for 3 hours as per recommendation in the method by British Pharmacopoeia in 1988. The extracted essential oil was dried with the use of anhydrous sodium sulfate and was kept in vials at 4°C before it will be tested by GC MS analysis. The total oils produced were calculated based on the dried weight of the plant materials.

Gas chromatography-mass spectrometry (GC-MS) is an analytical technique used by researchers with the help of an HP Agilent 2890, a gas chromatograph (GC) with an HP-55MS column, and a Quadrupole mass spectrometer at 70V. For 8 minutes, the column oven temperature was set at 60°C before it was increased to 250°C, having a rate of 2°C per minute. Helium is the carrier gas that flows through the column with 1 ml/min. Temperatures for the injector and detector were maintained at 250°C and 270°C, respectively. With the injection of 0.2 ml of the oil sample, the split ratio was set to 50:1. The NIST 02 and WILEY 7N libraries were used to compare retention periods with those of equivalent reference standards.

Moreover, published data was used to compare the retention index relative to n alkanes of the components. Then, the percentage compositions of the

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

various essential oils were determined based on the chromatographic peak areas.

Furthermore, the study utilized the process of determining a substance’s concentration and potency by its effect on living animals, in which in this case are the mosquito larvae. This analytical method is known as bioassays. Larvae that undergo ecdysis were exposed to four different concentrations of essential oil for 24 hours. To attain the final concentrations, the essential oil was diluted in 150 ml of filtered tap water after being dissolved in 1 ml of ethanol. The positive controlled variables were prepared by adding 1 ml of ethanol in 150 ml of water, while there were no additives on the negative ones. After the exposure period of mosquito larvae of 24 hours, they were collected and were placed in pure water after being rinsed with untreated water. The experiment was conducted with four replicates, each containing 20 larvae per concentration. Mortality rates of the larvae were recorded at 24, 48, and 72 hours after the treatment. Thereafter, the concentrations of both lethal and sub lethal (LC25, LC50 and LC90) were calculated along with the 95% confidence limits (CL) and slope of the concentration-mortality. The experiment's statistical analyses were carried out using a program called MINITAB. The findings are presented together with the subjects that were examined in each series while the mean and standard deviation (SD) are used to present data. The student's t test was used to determine the significance between the various

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

series. The probability value lower than 0.05 was chosen as the significance level.

Through hydro distillation, it was discovered that the aerial portions of Origanum vulgare contain a 1.74% pale-yellow liquid. Twenty-five components, or (99.86%) of the total identified essential oil, were identified by the GC MS analysis. Origanum vulgare's natural essential oil was revealed to be abundant in carvacrol (77.63%), while γ terpinene (6.79%) and cymene (5.41%) had lower concentrations. Another software called Graph Pad Prism 6 was used to determine the toxicity of the essential oil of Origanum vulgare to Culex pipiens larvae. The results of the study clearly showed that the increase in concentrations lead to an increase in the mortality rate of the larvae. After the treatment, the larvae, who are now intoxicated and incapacitated, exhibited a sudden shift in behavior as it sunk to the bottom of the container and died. Hence, Origanum vulgare Essential Oils exhibits a larvicidal activity against Culex pipiens

For hundreds of years, mosquitoes have been recognized as the fundamental arthropod vectors for the transmission of pathogens that kill

The Effect of Lemongrass Leaves and Stalks Extracts using Methanol as The Eco-friendly Larvicides on Fourth Instar Aedes aegypti Larvae

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

millions of people worldwide. Aedes aegypti are mosquito species responsible for chikungunya, dengue, and yellow fever. This family of mosquitoes are widely dispersed across tropical and subtropical areas.

Lemongrass is a tall, stalky plant that has traditionally been valued for its effectiveness as the key component in insecticides due to the citronella chemical compound present inside its stalk and leaves. A study by Mariam et al. (2021) utilized the essential oils extracted from lemongrass with the help of methanol to determine its larvicidal effect against Aedes aegypti larvae. In this study, the researchers obtained fourth instar larvae from the Faculty of Veterinary Universitas Syiah Kuala, Banda Aceh. Then, an 80:20 ratio of sterilized 40 mesh chick chow powder and yeast was used for its rearing in plastic containers. The treatments were replicated 4 times.

Three kilograms of the plant's leaves and stalks were crushed and extracted twice with ten liters or 90% methanol at room temperature in order to obtain the essential oils needed. After 24 hours, the extract was filtered and concentrated to dryness with the use of a rotary evaporator set at a temperature of 50°C. Then, 30 ml of the obtained pure extract was diluted using aquadest and stored at 4°C. Three extract concentrations were prepared using serial dilution of a stock extract: 2.5%, 5%, and 7.5%. 40 early fourth instar larvae were placed in each container with 50 ml of test solution. The positive control groups received three different concentrations with abate powder, whereas the negative control groups only received

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

aquadest. The conditions for detaining control and treated larvae were precisely the same. In the 2, 4, and 6 hours following treatment, the larvicidal activity was assessed three times. If the appendages did not move when a wooden dowel was inserted, the larva was considered to be dead.

On the other hand, there was no evidence of mortality in the negative control group. With the use of an analyze variance test, the mortality rate of each group was compared.

At the end of the experiment, the methanolic extract of Cymbopogon citratus was found to have a larvicidal effect on Aedes aegypti larvae. 100% mortality was recorded at 7.5% extract concentration, 95% mortality at 5% concentration, and 55% at 2.5%. Lethal concentrations (LC) of 50 and 90 were achieved at a concentration of 2.5% and 4.1%, respectively. Thus, concentrations at 2.5% and 4.1% were effective as larvicides.

Synthesis

As mosquitoes cause several diseases such as dengue and malaria which poses a threat to about 40% of the world’s population (CDC, 2019), humans began to acknowledge and utilize various strategies to deal with these pests. Particularly, the use of larvicides is practiced in order to eliminate mosquito larvae even before it develop into adults.

Plants have always been useful for humans. Humans specifically benefited from the essential oils of plants that were used for a variety of

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

purposes, one of which was for protection against mosquitoes. Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf are both widely used as primary ingredients of mosquito repellents and other pesticides due to their components that contribute to the elimination of mosquitoes.

A study by Mariam et al. (2021) utilized the extracts from the leaves and stalks of lemongrass obtained through methanol and was then used on Aedes aegypti mosquito larvae. Consequently, lemongrass extracts were proven to have larvicidal efficacy on mosquitoes. Additionally, a study by Bouguerra et al. in 2019 discovered that Oregano Essential Oils with carvacrol phenolic monoterpene (73.63%) as the major compound of the natural larvicide, can mitigate the resistance development of different mosquito species. With the use of hydro distillation as a method of oil extraction, their study established that Oregano Essential Oils possess larvicidal activity against mosquito larvae, specifically, C. pipiens larvae. The study at hand will utilize the method of hydro distillation in line with the process used in the study of Bouguerra et al. in 2019. However, the extraction process will be modified for it to be more feasible and efficient.

Culex mosquito larvae are characterized by their wide head and oval gill found at the bottom of their long antennae with large grass-like tips (Vork and Connelly, 2016). Culex larvae’sbodyisalso describedtobe“hairy”with a longer and lighter siphon than Aedes’ (Souza, 2017).

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

As commercially made larvicides pose numerous health and environmental risks, natural larvicides are safer to use. With that, the current study utilizes Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf and their essential oils as larvicides which promote a safer environment free from mosquitoes, specifically from Culex mosquito larvae.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

CHAPTER 3 METHODOLOGY

This chapter explains the research method or design that will be utilized for this study, the research samples, data gathering procedures, data analysis, and statistical treatment of data.

Research Method/Design

This research is a quantitative study that aims to gather quantitative data and conduct statistical analysis. To assess the efficacy of the synergistic larvicidal effect of Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf oil extract on Culex mosquito larvae, an experimental study will be conducted at the De La Salle University Dasmariñas laboratory.

As an experimental study, this research is primarily focused on uncovering the cause-and-effect relationship between variables by analyzing how different concentrations of the synergistic effect of Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf will affect the Culex mosquito larvae in terms of the time of effectivity and total mortality rate. The independent variable in this study is the varying Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf oil concentrations, the dependent variable is the time of effectivity and overall mortality rate, and lastly, the Culex mosquito larva, 1 ml of ethanol, and 150 ml of filtered tap water will

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

serve as the constant variable for the experiment. The experimental design will include three (3) treatments with two (2) concentrations each. The first and second treatment will employ simple oregano (T1) and lemongrass (T2) oil extracts, while the third treatment (T3) will use the combined Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf oil extracts. For every treatment, each concentration will comprise 25 and 50 ppm (25 and 50 mg/L) of solution containing simple or combined oil extracts, 1 ml of ethanol, and 150 ml of filtered tap water. Furthermore, 120 Culex mosquito larvae will be divided into six (6) groups of 20 larvae. Each group will administer the corresponding amount and concentration of simple and combined Origanum vulgare L. and Cymbopogon citratus (DC.) Stapf oil extracts. To examine the differences and effectiveness of the larvicides, several tests, including the total mortality rate test, time of effectivity test, and t test, will be conducted on the three treatments.

Data Gathering Procedures

Figure 2. Flow chart of the Data Gathering Procedures.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Phase I. Collecting of Origanum vulgare L., Cymbopogon citratus (DC.) Stapf, and Culex Mosquito Larvae. This research will conduct an experiment to create the most effective naturally made larvicide. The researchers will primarily focus on preparing for the experiment by reserving the laboratory and by gathering several measuring materials, different apparatus, ingredients for different concentrations, and the larvae.

The laboratory will be used to conduct the extraction of essential oil as well as the preparation of different concentrations (of/for naturally made larvicides). A reservation form must be filled out and submitted days prior to the actual use of the laboratory. Measuring materials such as beakers, flasks and weighing scales will be needed for the accurate measurement and analysis. Additionally, a stirring rod for stirring and separate containers for wet and dry ingredients such as ethanol, dried oregano, lemongrass, and distilled water are also needed. For the extraction of oil, Clevenger apparatus will be used.

The researchers will request and gather 120 Culex mosquito larvae from UPLB, already divided into six (6) groups, containing 20 mosquito larvae each. The mosquito larvae will be fed with biscuit-dried yeast with a ratio of 75:25 by weight.

Phase II. Extraction of Essential Oil using Hydro Distillation. After gathering the materials needed, the group’s chosen method of

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

essential oil extraction is hydro distillation. For the simple oil extracts, the gathered 100 g dried leaves of the plant will be cut into small pieces before they are mixed and soaked in a container of distilled water. To obtain the combined essential oil, ingredients will be equally divided into two. The researchers will only use 50 g of oregano and 50 g of lemongrass, resulting in a 100 g of combined plants. Using the Clevenger apparatus, the heater will be turned on and it will be heated at 90°C to 100°C until it starts to boil. Once the boiling starts, the temperature will be lowered to 40°C to 50°C which will be left for 3 to 4 hours. As the boiling process takes place, the condensing apparatus collects both steam and oil vapor. The excess water from the resulting mixture is then forced out of a hole in the apparatus, leaving the distillate in the receiving flask as the finished product. Then, the oil will be collected in a vial for use.

Phase III. Making of the Simple Oil Extract and the Combined Essential Oil. After extracting the oil from the plants, the researchers will prepare the apparatuses and chemicals needed to create the desired concentrations. The group will prepare 2 different concentrations each for lemongrass simple oil extract and oregano oil extract with 25 and 50 ppm (25 and 50 mg/L) of the solution with the study of Bouguerra et al. (2019) as the basis. Furthermore, the researchers will utilize 25 mg/L and 50 mg/L concentrations for the preparation of the combined essential oil. The extracted essential oil will be dissolved in 1 ml ethanol. Then, it will be

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

diluted in 150 ml of filtered tap water. The formula indicated below will help the researchers prepare the solution that will then be used for the experiment.

������= ������������������������(��) ��������������������������(��) × 106

1 ������ = 1 ����/��

Phase IV. Experimentation and Observation. The 120 Culex mosquito larvae will be divided into six (6) groups consisting of twenty (20) larvae per container. Each group of larvae will be exposed to 25 mg/L and 50 mg/L concentrations of the essential oils. The researchers will observe the larvae every 2 minutes if there are deaths and record the time of effectivity in minutes before solving for the average time of effectivity. The researchers will also record the total mortality rate after an hour for a day.

Phase V. Data analysis. After collecting the data, the researchers will analyze, tabulate, organize, and interpret the gathered information to determine the synergistic effect of the Cymbopogon citratus (DC.) Stapf and Origanum vulgare L.

Statistical Treatment of Data

All data will be gathered through experimentation and observation. Bioassays, specifically the time of effectivity test and the total mortality test, will be used to determine the synergistic effect of Origanum vulgare L. and

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Cymbopogon citratus (DC.) Staph in Culex mosquito larvae in terms of total mortality rate. Moreover, the researchers will determine if there is a significant difference between the total mortality rate of the Culex mosquito larvae and the time of effectivity after application of the different concentrations of the lemongrass and oregano simple oil extract to the combined oil extract using Student’s t test.

Time of Effectivity Test. The time of effectivity test will be conducted through observation. A camera will be prepared and used to help document the time of death of a larva and the average time of effectivity in minutes. Through observation, the researchers will record the number of larvae that sunk at the bottom of the container in an interval of 2 minutes for an hour. Afterwards, the average time of effectivity in minutes will be recorded per group of larvae.

Total Mortality Rate Test. The total mortality rate test will be conducted after gathering the required data. The researchers will prepare and set up a camera to document the larvicidal activity of each concentration of the simple and combined Cymbopogon citratus and Origanum Vulgare L. oil extracts. The number of deaths of the mosquito larvae will be observed and recorded every two minutes for an hour. Following that, the mortality rate will be calculated by dividing the number of dead mosquito larvae by the total number of larvae exposed to a specific

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

concentration of a specific extract multiplied by 100%. The formula from the study of Martianasari R. and Hamid P.H. (2019) indicated below will be used to help the researchers compute the total mortality rate. ������������������(%) = ( ���������������������������������������������������� ����������������������������������������������������������������������) × 100% T-test. The data collected from the experiment will be analyzed using Student’s t test in Microsoft Excel. The combined essential oil treatments will be compared individually to the simple oil extract in terms of total mortality rate and time of effectivity. All statistical analysis will be done at 5% probability level.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

REFERENCES

Abubakar A, Haque M. 2020. Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. Journal of Pharmacy And Bioallied Sciences. 12(1):1. doi:10.4103/jpbs.jpbs_175_19. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398001/.

Abutaha N, Al Mekhlafi FA, Al Keridis LA, Farooq M, Nasr FA, Al Wadaan M. 2018 Mar 26. Larvicidal potency of selected xerophytic plant extracts onCulex pipiens(Diptera: Culicidae). Entomological Research. doi:10.1111/1748-5967.12293.

Advocacy & Management Group, Inc. (AMCA). “Biology - American Mosquito Control Association.” Mosquito.org, 2019, www.mosquito.org/page/biology.

Alayo, Muinat & Femi Oyewo, M. & Bakre, Lateef & Fashina, A.. (2015). LARVICIDAL POTENTIAL AND MOSQUITO REPELLENT

ACTIVITY OF CASSIA MIMOSOIDES EXTRACTS. The Southeast Asian journal of tropical medicine and public health. 46. 596-601.

Alcalay Y, Puzhevsky D, Tsurim I, Scharf I, Ovadia O. 2018. Interactive and sex‐specific life‐history responses of Culex pipiens mosquito larvae to multiple environmental factors. Journal of Zoology. 306(4):268 278. doi:10.1111/jzo.12611.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Alhassan, M., Lawal, A., Nasiru, Y., Suleiman, M., Sa ya, A.M., Bello, N. (2018). Extraction and formulation of perfume from locally available lemongrass leaves. ChemSearch Journal, 9(2),40 44.

Almusaed A, Al-Samaraee SMS. 2017. Grasses: Benefits, Diversities and Functional Roles. BoD Books on Demand. [accessed 2022 Oct 2].

6llfDKm1&sig=Y4

Amusan B, Ogbogu S. 2020. Surveillance of mosquito larvae (Diptera: Culicidae) in microhabitats of a University Campus in Southwestern Nigeria. UNED Research Journal / Cuadernos de Investigación

Aranda C. 2017. The Common House Mosquito: An Unwanted Companion

Our Lives - Blog. ISGlobal.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Araonu CF, Ike AC, Ngwu GI. 2018. Larvicidal effects of crude methanolic and n hexane extracts of Cymbopogon citratus and Ocimum gratissimum on Anopheles gambiae mosquito. Nigerian Journal of Parasitology. 39(1):30. doi:10.4314/njpar.v39i1.6

Auerswald H, Ruget AS, Ladreyt H, In S, Mao S, Sorn S, Tum S, Duong V, Dussart P, Cappelle J, Chevalier V. Serological evidence for Japanese encephalitis and West Nile virus infections in domestic birds in Cambodia. Frontiers in veterinary science. 2020 Jan 29;7:15. Barba M, Fairbanks EL, Daly JM. Equine viral encephalitis: prevalence, impact, and management strategies. Veterinary Medicine: Research and Reports. 2019;10:99.

Batti ACS, Younoussa L, Nukenine EN, Oumarou MK, Fomena A. 2019 Jun

4. Larvicidal Activity of Two Cymbopogon Species Leaf Extracts and Essential Oils against Anopheles gambiae Gilles (Diptera: Culicidae). Journal of Applied Life Sciences International.:1 12. doi:10.9734/jalsi/2019/v21i130096

Bhattacharya, Sajal & Basu, Probal. (2016). The Southern House Mosquito, Culex quinquefasciatus: profile of a smart vector. 73-81.

Bellassoued K, Ben Hsouna A, Athmouni K, van Pelt J, Makni Ayadi F, Rebai T, Elfeki A. 2018. Protective effects of Mentha piperita L. leaf essential oil against CCl4 induced hepatic oxidative damage and

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

renal failure in rats. Lipids in Health and Disease. 17(1). doi:10.1186/s12944 017 0645 9.

Bellone R, Failloux AB. The role of temperature in shaping mosquito borne viruses transmission. Frontiers in Microbiology. 2020 Sep 25;11:584846.

Bouguerra N., Fouzia T.D., Soltani N. 2019. Oregano Essential Oil as Potential Mosquito Larvicides (PDF). Research Gate. https://www.researchgate.net/publication/333677930_Orega no_Essential_Oil_as_Potential_Mosquito_Larvicides

Brügger BP, Martínez LC, Plata-Rueda A, Castro BM de C e, Soares MA, Wilcken CF, Carvalho AG, Serrão JE, Zanuncio JC. 2019. Bioactivity

of the Cymbopogon citratus (Poaceae) essential oil and its terpenoid constituents on the predatory bug, Podisus nigrispinus (Heteroptera: Pentatomidae). Scientific Reports. 9(1):8358. doi:10.1038/s41598 019 44709 y. https://www.nature.com/articles/s41598 019 44709 y

CDC. 2020 Mar 9. Control During an Outbreak | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/mosquitoes/mosquitocontrol/community/mosquito-control-during-outbreak.html.

CDC. 2020 Mar 6. Culex Mosquito Life Cycle | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/mosquitoes/about/life cycles/culex.html

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

CDC. 2020 Aug 24. Larvicides | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/mosquitoes/mosquito control/athome/outside your home/larvicides.html

CDC. 2020 Mar 9. Larvicides | CDC. Centers for Disease Control and Prevention. https://www.cdc.gov/mosquitoes/mosquitocontrol/community/larvicides.html.

Chan CA, Ho LY, Sit NW. 2022. Larvicidal Activity and Phytochemical ProfilingofSweetBasil(OcimumbasilicumL.)LeafExtractagainst AsianTigerMosquito(Aedesalbopictus).Horticulturae.8(5):443. doi:10.3390/horticulturae8050443.

County of San Diego Communications Office. “Know Your Mosquitoes?

Read the Tale of the Tape!” San Diego County News Center,13 Oct. 2016, www.countynewscenter.com/know your mosquitoes read the tale of the tape/.

Department of Molecular Virology and Microbiology. “Mosquito Borne Diseases.” Baylor College of Medicine, 2017, www.bcm.edu/departments/molecular-virology-andmicrobiology/emerging-infections-and-biodefense/mosquitoes.

Desai MA, Parikh J. 2015. Extraction of Essential Oil from Leaves of Lemongrass Using Microwave Radiation: Optimization,

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Comparative, Kinetic, and Biological Studies. ACS Sustainable Chemistry & Engineering. 3(3):421 431. doi:10.1021/sc500562a.

de Oliveira AA, França LP, Ramos A de S, Ferreira JLP, Maria ACB, Oliveira KMT, Jr ESAraújo, da Silva JN, Branches ADS, Barros G de A, et al. 2021. Larvicidal, adulticidal and repellent activities against Aedes aegypti L. of two commonly used spices, Origanum vulgare L. and Thymus vulgaris L. South African Journal of Botany. 140:17 24. doi:10.1016/j.sajb.2021.03.005.

ElKasemBoslyHA.2022.Larvicidalandadulticidalactivityofessential oilsfromplantsoftheLamiaceaefamilyagainsttheWestNilevirus vector, Culex pipiens (Diptera: Culicidae). Saudi Journal of Biological Sciences. 29(8):103350. doi:10.1016/j.sjbs.2022.103350.

El Husseiny I, Elbrense H, Roeder T, El Kholy S. 2018. Hormonal modulation of cannibalistic behaviors in mosquito (Culex pipiens) larvae. Journal of Insect Physiology. 109:144–148. doi:10.1016/j.jinsphys.2018.08.001.

Elyemni M, Louaste B, Nechad I, Elkamli T, Bouia A, Taleb M, Chaouch M, Eloutassi N. 2019 Apr 1. Extraction of Essential Oils of Rosmarinus officinalis L. by Two Different Methods: Hydrodistillation and

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Microwave Assisted Hydrodistillation. The Scientific World Journal. https://www.hindawi.com/journals/tswj/2019/3659432/

Goselle O, Gyang D, Adara O, Effiong K, Nanvyat N, Adulugba I, Kumbak D, Ahmadu Y, Mafuyai H. 2017. A comparative Study of the Larvicidal Activity of Lemongrass (Cymbopogan citratus) from Different Methods of Extraction. Journal of Academia and Industrial Research (JAIR). 6(3). [accessed 2022 Oct 2]. https://irepos.unijos.edu.ng/jspui/bitstream/123456789/2240/1/Gose lle%203.pdf

Govindarajan M, Rajeswary M, Hoti SL, Benelli G. 2016. Larvicidal potential of carvacrol and terpinen-4-ol from the essential oil of Origanum vulgare (Lamiaceae) against Anopheles stephensi, Anopheles subpictus, Culex quinquefasciatus and Culex tritaeniorhynchus (Diptera: Culicidae). Research in Veterinary Science. 104:77 82. doi:10.1016/j.rvsc.2015.11.011.

HrnˇciˇcMK, Cör D, Simonovska J, Knez Željko, Kavrakovski Z, Rafajlovska V. 2020. Extraction Techniques and Analytical Methods for Characterization. Multidisciplinary Digital Publishing Institute (MDPI). https://www.mdpi.com/1420-3049/25/20/4735/pdf-vor Insecticide Poisoning Injuries and Poisoning. MSD Manual Consumer Version. https://www.msdmanuals.com/home/injuries and poisoning/poisoning/insecticide poisoning

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

International Federation of Red Cross and Red Crescent Societies. 2019. Information bulletin Philippines: Dengue Outbreak. https://www.ifrc.org/docs/Appeals/19/IBPHdg190719.pdf

IqbalA,QureshiNA,AlhewairiniSS,ShaheenN,HamidA,QureshiMZ. 2022.Biocidalaction,characterization,andmoleculardockingof Mentha piperita (Lamiaceae) leaves extract against Culex quinquefasciatus(Diptera:Culicidae)larvae.HanoC,editor.PLOS ONE.17(7):e0270219.doi:10.1371/journal.pone.0270219.

Kumar V., Marković T., Emerald M., Dey A. (2016) Herbs: Composition and Dietary Importance. Encyclopedia of Food and Health. Academic Press. Pages 332 337. https://doi.org/10.1016/B978 0 12 384947 2.00376 7.

Linthicum KJ, Britch SC, Anyamba A. Rift Valley fever: an emerging mosquito borne disease. Annu Rev Entomol. 2016 Mar 11;61(1):395 415. Majewska, E., Kozłowska, M., Gruczyńska-Sękowska, E., Kowalska, D., Tarnowska, K. (2019). Lemongrass (Cymbopogon citratus) Essential Oil: Extraction, Composition, Bioactivity and Uses for Food Preservation a Review. Polish Journal of Food and Nutrition Sciences, 69(4), 327 341. https://doi.org/10.31883/pjfns/113152

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Mariam T, Oktiviyari A, Harahap AY. The Effect of Lemongrass Leaves and Stalks Extracts using Methanol as The Eco friendly Larvicides on Fourth Instar Aedes aegypti Larvae | Open Access Macedonian Journal of Medical Sciences. 2021 Oct 4. oamjmseu. https://oamjms.eu/index.php/mjms/article/view/6727.

MartianasariR,HamidPH.2019.Larvicidal,adulticidal,andoviposition deterrentactivityofPiperbetleL.essentialoiltoAedesaegypti. March2019. 12(3):367 371. doi:10.14202/vetworld.2019.367 371.

Mosquito Borne Diseases. 2017. Baylor College of Medicine. https://www.bcm.edu/departments/molecular-virology-andmicrobiology/emerging-infections-and-biodefense/mosquitoes.

Mosquito Larvae. Missouri Department of Conservation. https://mdc.mo.gov/discover nature/field guide/mosquito larvae

Mukarram M, Choudhary S, Khan MA, Poltronieri P, Khan MMA, Ali J, Kurjak D, Shahid M. 2021 Jun 21. Lemongrass Essential Oil Components with Antimicrobial and Anticancer Activities. wwwpreprintsorg. doi:10.20944/preprints202106.0500.v1. https://www.preprints.org/manuscript/202106.0500/v1.

Muthukumar P, Suvarna V P, Poovizhi K V. 2021 Aug 25. Assessment of larvicidal properties of bio pesticide (Cymbopogon citratus) plant

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

extracts against culex mosquitoes larva. ResearchGate. https://www.researchgate.net/publication/354202171_Assessment_ of_larvicidal_properties_of_bio pesticide_Cymbopogon_citratus_plant_extracts_against_culex_mo squitoes_larva

Nanju GO, Gyang D, Adara O, Effiong K, Nanvyat N, Barshepn Y, Matur B, Kumbak D, Ahmadu Y, Mafuyai H. 2018. A comparative study of the growth pattern of the larvae of Culex Linnaeus, 1758 in various water bodies and the implications for environmental control. MOJ Ecology & Environmental Sciences. Volume 3(Issue 3). doi:10.15406/mojes.2018.03.00084. [accessed 2022 Oct 2]. https://medcraveonline.com/MOJES/a-comparative-study-of-thegrowth pattern of the larvae of culex linnaeusnbsp1758 in various water bodies and the implications for environmental control.htm

National Pesticide Information Center. 2019. Insecticides. Orstedu. http://npic.orst.edu/ingred/ptype/insecticide.html

Njouku-Tony RF, Ebe TE, Mgbemena I, Chinedu I. 2015 Feb. Larvicidal Effect of Cymbopogon Citratus root and leaf on the first instar laval stage of Anopheles gambiae, culex quinquefasciatus and Aedes eagypti. ResearchGate. https://www.researchgate.net/publication/325498255_Larvicidal_Eff ect_of_Cymbopogon_Citratus_root_and_leaf_on_the_first_instar_l

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

aval_stage_of_Anopheles_gambiae_culex_quinquefasciatus_and_ Aedes_eagypti

Ocimum basilicum L. 2022 Feb 23. National Parks. https://www.nparks.gov.sg/florafaunaweb/flora/2/2/2274.

O’Malley G, O’Malley R. 2022 Sep. Insecticide Poisoning. MSD Manual Consumer Version. https://www.msdmanuals.com/home/injuriesand poisoning/poisoning/insecticide-poisoning.

Omolade OO, Adetutu SA. 2018. Oviposition and Breeding Water Sites Preferences of Mosquitoes within Ojo area, Lagos State, Nigeria. Biomedical Journal of Scientific & Technical Research. 7(5). doi:10.26717/bjstr.2018.07.001565

Oregano (Origanum vulgare). Restorative Medicine. https://restorativemedicine.org/library/monographs/oregano/ Paramasivan R, Mishra AC, Mourya DT. West Nile virus: the Indian scenario. Indian Journal of Medical Research. 2003;118:101-8. Reddy BS, Mohana AK, Sam Jiju A, Fernandez V. 2018 Apr. The larvicidal effect of lemongrass (Cymbopogon citratus) leaf extract on Aedes mosquito larvae. wwwherdinph. [accessed 2022 Oct 2].

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

https://www.herdin.ph/index.php/herdin home?view=research&cid=72162

Repellents American Mosquito Control Association. 2019. Mosquitoorg. https://www.mosquito.org/page/repellents.

Rudayni HA, Basher NS, AL-keridis LA, Ibrahim NAdam, Abdelmageed E. 2021. The Efficiency of Ethanolic Extract of Ocimum basilicum Leaves and Flowers agiainst Mosquito Larvae. Entomology and Applied Science Letters. 8(3):46 53. doi:10.51847/5wpmv7xyxl.

Ruiz Santiago N, Granados Echegoyen CA, Pérez Pacheco R, Montesinos G, Platzer EG, Rodríguez-Ortiz G. 2018. Effects of Salinity and pH on the Infective Capacity of Romanomermis iyengari and Romanomermis culicivorax in Larvae of Culex quinquefasciatus Mosquito. Southwestern Entomologist. 43(3):625 633. doi:10.3958/059.043.0307.

Seye F, Fall A, Toure M, Ndione RD. 2021 Mar. Histopathological Effects of Cymbopogon citratus (Lemongrass) Essential Oil on Late Third Instar Larvae of Aedes aegypti L. (Diptera: Culicidae). ResearchGate. https://www.researchgate.net/publication/350100260_Histopatholog ical_Effects_of_Cymbopogon_citratus_Lemongrass_Essential_Oil_ on_Late_Third_Instar_Larvae_of_Aedes_aegypti_L_Diptera_Culici dae

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Smith LB, Kasai S, Scott JG. 2016. Pyrethroid resistance in Aedes aegypti and Aedes albopictus: Important mosquito vectors of human diseases. Pesticide Biochemistry and Physiology. 133:1 12. doi:10.1016/j.pestbp.2016.03.005.

Souto AL, Sylvestre M, Tölke ED, Tavares JF, Barbosa-Filho JM, CebriánTorrejón G. 2021. Plant Derived Pesticides as an Alternative to Pest Management and Sustainable Agricultural Production: Prospects, Applications and Challenges. Molecules. 26(16):4835. doi:10.3390/molecules26164835. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8400533/

Souza T. 2017. How to identify Culex, Anopheles and Aedes mosquitoes and their larvae? ResearchGate. https://www.researchgate.net/post/How_to_identify_Culex_Anophel es_and_Aedes_mosquitoes_and_their_larvae#:~:text=Aedes%20a nd%20Culex%20are%20very

Suryawanshi, M.A., Mane, V.B., Kumbhar, G.G. (2016). Methodology to extract essential oils from lemongrass: solvent extraction approach. International Research Journal of Engineering and Technology, 3(8), 1775-1780 .

Valent BioSciences. “Culex Public Health.” www.valentbiosciences.com. 2017.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

https://www.valentbiosciences.com/publichealth/insects/mosquitoes /culex/.

Vork D, Connelly R. 2016 Jun. a mosquito Culex (Melanoconion) pilosus. entnemdeptufledu. [accessed 2022 Oct 2]. https://entnemdept.ufl.edu/creatures/misc/flies/culex_pilosus.htm?f bclid=IwAR2cy_iW7a13AMivp7ZuxjoxcgU576CvPnx4eouCO1Hm6r fsNrS8tlCsowk.

Wagh, Akash & Jaiswal, Swapnil & Bornare, Deepak. (2021). A review: Extraction of essential oil from lemongrass as a preservative for animal products. Journal of Pharmacognosy and Phytochemistry. 10. 26-31. 10.22271/phyto.2021.v10.i3Sa.14187.

Waris M, Nasir S, Abbas S, Azeem M, Ahmad B, Khan NA, Hussain B, AlGhanim KA, Al Misned F, Mulahim N, et al. 2020. Evaluation of larvicidal efficacy of Ricinus communis (Castor) and synthesized green silver nanoparticles against Aedes aegypti L. Saudi Journal of Biological Sciences. 27(9):2403 2409. doi:10.1016/j.sjbs.2020.04.025.

Wilkerson RC, Linton Y-M, Strickman D. 2021. Mosquitoes of the world 1. Baltimore, Maryland Johns Hopkins University Press.

World Health Organization. 2018 Jul 20. Zika virus. Whoint. https://www.who.int/news room/fact sheets/detail/zika virus.

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL SCIENCE, TECHNOLOGY, ENGINEERING AND MATHEMATICS

Yadav, Ishwar & Devi, Ningombam. (2017). Pesticides Classification and Its Impact on Human and Environment. Yuanpeng H., Jingyi L., Lei S. 2021. A Carvacrol Rich Essential Oil Extracted From Oregano (Origanum vulgare “Hot & Spicy”) Exerts Potent Antibacterial Effects Against Staphylococcus aureus. Frontiers in Microbiology Volume 12. 10.3389/fmicb.2021.741861. https://www.frontiersin.org/articles/10.3389/fmicb.2021.741861.

Dasmariñas

De La Salle University Dasmariñas

SENIOR HIGHSCHOOL

TECHNOLOGY,

APPENDIX B

AND MATHEMATICS

BUDGETARY REQUIREMENTS

Items Amount Total

Biscuit 50/pack PhP 50 Ethanol 110/500 mL 110

Lemongrass 105/100 g 210 Oregano 100/100 g 200 Pipettes 20/piece 140

Specimen Sterile Container 30/piece 210

Surgical Gloves 450/box 450 Vial 60/3 dram 180 Yeast 75/80 g 75

Total PhP 1,625.00