CHAPTER 1
INTRODUCTION

I. Background of the Study
Mosquitoes are insects in the order Diptera. They usually breed in transient water sources, such as flooded areas, snowpools, and ditches (Rutgers [date unknown]). A mosquito goes through four stages: egg, larva, pupa, and adult. Most eggs hatch into larvae within 48 hours (Biology Notes… 1983).
Mosquitoes are vectors of numerous diseases. Aedes aegypti, a domestic mosquito living in close association with humans, is the primary vector of yellow fever and is also a potential vector of dog heartworm, Murray Valley encephalitis and Ross River viruses (Zettel and Kauffman 2008; Russell 1996). Moreover, it is the principal vector of dengue viruses in tropical and subtropical regions throughout the world (Luz and others 2008). The dengue viruses acquired from the said species are known to cause an estimated 50 million human infections annually (Olson and others [date unknown]). Dengue cases in Iloilo province has been reported to reach 4,825 with 27 deaths from January 1 to September 18, 2010 (The News Today 2010).
Humans acquire the diseases carried by Ae. aegypti through mosquito bites. Since there is no effective vaccine for the control of these diseases, prevention of these bites is used as an alternative control (Tawatsin and others 2006). Furthermore, the control of these diseases depends on reduction and elimination of Ae. aegypti (Olson and others [date unknown]). Nowadays, the Philippine government relies on direct applications of aerosol insecticides, fogging, and ovitraps to prevent Aedes aegypti proliferation (Official Gazette [date unknown]; Philippine Daily Inquirer 2010).
Aerosol insecticides are available in handy canister form and are simply sprayed (Olson and others [date unknown]). In most countries in Southeast Asia, mosquito breeding sites are sprayed with dichlorodiphenyltrichloroethane or DDT (Scanlon [date unknown]). Fogging, or space spraying, on the other hand, disperses hundreds of millions of tiny droplets less than 50 μm in diameter into the air. Its efficacy only lasts until the droplets remain airborne (Slideshare 2008). An ovitrap, developed by the Department of Science and Technology, is a black tin can with a piece of lawanit wood stuck inside with a black “organic” solution or chemical developed by epidemiologists. Dengue-carrying mosquitoes gather inside the black container and lay their eggs but the larvae end up being killed in the can (Leyte Samar Daily Express 2010).
On the downside, the outdoor application of aerosol insecticides is costly and ineffective, since the majority of female Ae. aegypti rest indoors to avoid insecticide contact. Moreover, many insecticides are useless due to the spread of resistant mosquitoes (Olson and others [date unknown]). Spraying, although inexpensive and obtainable, enhances resistance of immature stages of mosquitoes to pesticides (Scanlon [date unknown]). Meanwhile, fogging kills adult mosquitoes but not hatchlings. It also causes mosquitoes to flee, transfer to other areas, and become immune to the chemicals used (The Star 2010; Official Gazette [date unknown]).
Another way to effectively decrease the number of diseases transmitted by mosquitoes is through the elimination of mosquito larvae and ova. This said elimination can be done through the use of larvicides and ovicides, which can be chemicals or natural bacteria (Kempraj and Bhat 2008). Killing mosquito ova and larvae before they emerge as adults can reduce or eliminate the need for ground or aerial application of pesticides to kill adult mosquitoes. Currently, larvicides such as Bacillus sphaericus, Methoprene and Temephos are being used. However, in the use of these larvicides, there is an acute and chronic risk to some fish and freshwater invertebrate species. In large doses, these larvicides can cause nausea or dizziness (US EPA 2010; DOHMH 2010). Extracts from neem and Cyperus plants have ovicidal properties. However, they have strong and pungent smells that can cause irritation or nausea (Kempraj and Bhat 2008; Suite101 [date unknown]). Ovitraps such as grass infusions and cyfluthrin, on the other hand, are moderately toxic to mammals and are plain toxic to fish (Singh and Bansal 2005; Extoxnet 2010).
Crude extracts from certain plants have been proven to be effective in killing larvae and ova of mosquitoes. Extracts obtained from Solanum xanthocarpum were proven to be effective biological mosquito larvicides against Culex, while extracts obtained from the fruits of Momordica charantia were found to be larvicidal against Culex, Anopheles and Aedes (Changbungjong and others [date unknown]; Singh and others 2006). Crude extracts from Calodendrum capense were discovered to be larvicidal against Aedes, as well (Kiprop and others 2005).
Other crude extracts were also proven to exhibit ovicidal properties against certain genera of mosquitoes. Crude extracts from Ervatamia coronaria and Caesalpinia pulcherrima are known to be excellent ovicides against Culex, Aedes and Anopheles mosquitoes (Govindarajan and others 2010).
Crude extracts from guava, a tropical fruit common in the Philippines that is known for its medical purposes as an antiseptic, an antioxidant, and an antibacterial (Production Guide on Guava [date unknown]), are known to contain the following sulfur volatiles: limonene, (Z,E)-a-farnesene, (E,E)-a-farnesene, b-farnesene, curcumene, a-pinene, hexanal, hydrogen sulfide, sulfur dioxide, methanethiol, dimethyl sulfide, dimethyl disulfide, methional, and dimethyl trisulfide. These sulfur volatiles are also found in the essential oil of neem, which is considered to be larvicidal and ovicidal to Aedes aegypti (Rouseff and others 2008).
An aqueous extract, on the other hand, is a water-based preparation of a plant substance containing its biologically active portion without the cellular residue (The Free Dictionary 2011). Aqueous extracts obtained from Psidium guajava L. leaves are known to be composed of tannins, β-sitosterol, maslinic acid, essential oils, triterpenoids, saponins and flavonoids (Osman and others 1974; Arima and Danno, 2002; Begum and others 2004; Abreu and others 2006). Triterpenoid saponin, a major component of the said extract, targets and depresses acetylcholinesterase activity in mosquito larvae, thereby killing them (Tandon and Sirohi 2010).
The researchers determined if aqueous and crude extracts from guava leaves of the different concentrations (0, 50, 75, 100, 1000 and 2000 ppm) exhibit larvicidal and ovicidal properties against Aedes aegypti.

II. Statement of the Problem
This study evaluated the ovicidal and larvicidal properties of guava (Psidium guajava L.) crude extract of different concentrations (0, 50, 75, 100, 1000 and 2000 ppm) and aqueous extracts from guava leaves against Aedes aegypti by determining the hatch percentage, percent larval mortality and lethal concentration after seventy-two (72) hours of continuous exposure.

III. Objectives
This study aimed to evaluate the ovicidal and larvicidal properties of crude and aqueous extracts of Psidium guajava L. leaves against Aedes aegypti L. by:
A.1. testing the Psidium guajava crude extract of different concentrations (0, 50, 75, 100, 1000 and 2000 ppm) and aqueous extract against Aedes aegypti ova and;
A.2. testing the Psidium guajava crude extract of different concentrations (0, 50, 75, 100, 1000 and 2000 ppm) and aqueous extract against Aedes aegypti larvae.

IV. Significance of the Study
Prevention of mosquito bites through reduction and elimination of Ae. aegypti is vital. The crude and aqueous extract obtained from guava (Psidium guajava), a plant that is abundant in tropical regions like the Philippines, contain properties that make it a potential larvicide/ovicide that could kill mosquito larvae/ova.
The essential oils found in the crude extract are synergistic. The chemical components found in them complement each other. Thus, when blended together, their effectivity becomes greater compared to when they are used singly (Suite 101 2008). This study introduced a new method of mosquito control that is both convenient and inexpensive. This study will primarily benefit those who live in regions where the mosquito Aedes aegypti is abundant.

V. Scope and Delimitations of the Study
The study only dealt with the larvicidal and ovicidal properties of the crude and aqueous from guava leaves. Other properties such as adulticidal and mosquito-deterrence were not included.
This study only dealt with the larvae of mosquito species Aedes aegypti, which were obtained from the Research Institute of Tropical Medicine (RITM). The larvae were cultured in the institute’s laboratory. Additionally, seven hundred fifty (750) eggs obtained from the same laboratory were used in the ovicidal test.
This study only used crude and aqueous extract obtained from the leaves of guava. It did not include extracts obtained from the stems, roots, or from other parts of the tree. Guava (Psidium guajava) leaves were randomly collected from Brgy. Tubungan, Barotac Nuevo, Iloilo. The extraction of guava crude extract was done using the solvent extraction method and was conducted in the University of San Agustin – Chemistry Laboratory. The aqueous extraction, on the other hand, was conducted in one of the researchers’ residence in Barotac Nuevo, Iloilo. This study did not include the characterization of the water used in ovicidal and larvicidal tests.
The procedure for the entire experiment to evaluate the ovicidal and larvicidal properties of guava crude and aqueous extract was adapted from the guidelines provided by the Research Institute of Tropical Medicine. In this study, probit analysis was performed since the parameters it measures (Lethal Concentration and Lower and Upper Confidence Limits) are needed in attaining the objectives of the study. This study was conducted in April to May 2011.

VI. Definition of Terms

Aedes aegypti – It is a domestic mosquito that lives in close association with humans, breeding in artificial containers around houses and is responsible for the majority of dengue and yellow fever transmission (Mosquito Age [date unknown]; Princeton 2010).
In this study, Aedes aegypti will be the mosquito species used in testing the larvicidal and ovicidal properties of guava crude and aqueous extract.
Aqueous Extract – It is a water-based preparation of a plant or animal substance containing the biologically-active portion of the plant or substance without its cellular residue (The Free Dictionary [date unknown]).
In this study, the ovicidal and larvicidal properties of the aqueous extract of guava leaves were tested.
Crude Extract – It is a concentrate obtained by treating a crude material, such as plant or animal tissue, with a solvent, evaporating part or all of the solvent from the resulting solution, and standardizing the resulting product (Mosby’s Dental Dictionary 2008). In this study, the larvicidal and ovicidal properties of the crude extract of guava were evaluated.
Essential Oil – It is a highly volatile and aromatic liquid obtained through extraction. Generally, it constitutes the odorous principles of a plant, having the taste or smell of the original botanical used (Shaveinfo [date unknown]).
In this study, essential oil is contained in the crude extract. The essential oil contains the sulfur volatiles, which made the crude extract a potential larvicide and ovicide.
Guava – It is a small tropical shrubby tree that bears a fruit with yellow skin and pink pulp (Princeton 2010). In this study, the ovicidal and larvicidal properties of the crude and aqueous extract from guava leaves were tested.
Hatch percentage – It is the percentage of eggs that hatched (Websters Online [date unknown]). In this study, the hatch percentage was used to determine the effectivity of the guava crude and aqueous extract.
Larva – It is the immature free-living form of most invertebrates and amphibians and fish which at hatching from the egg is fundamentally unlike its parent and must, therefore, metamorphose (Princeton 2010). In this study, Aedes aegypti larvae were used in testing the larvicidal property of guava crude and aqueous extract.
Larvicide – It is a biological or chemical product from specific bacteria that is lethal to mosquito larvae but not to other organisms (Fight the Bite 2010)
Lethal concentration – It is the amount of a drug or other agent that if administered to an animal or human will prove fatal (The Free Dictionary 2011). In this study, the LC50 and LC99 of the crude extract obtained from guava were measured using Probit Analysis.
Ovicide – It is a material which kills the egg stage of an organism
(Bacheler [date unknown]).
Percent larval mortality – It is the ratio of deaths in an area to the population of that area (WordNet 3.0 [date unknown]). In this study, the percent larval mortality was measured after seventy-two (72) hours of treatment.
Stimulus – It is any information coming into the body that is capable of generating a nerve impulse (National Institutes of Health [date unknown]). In this study, a needle was used as the stimulus. A larva will be considered dead if it cannot be induced to move when probed with the needle in the siphon or cervical region.