Larvicidal Effect of Used Coffee Grounds ...

CHAPTER I

INTRODUCTION

Vector-borne diseases are an ongoing public health concern in which it can be

transmitted by a bite of infected arthropod species, such as mosquitoes, ticks, triatomine

bugs, sandflies and blackflies. Every year there are more than 1 billion cases and over 1

million deaths from vector-borne diseases such as malaria, dengue, schistosomiasis,

human African trypanosomiasis, leishmaniasis, Chagas disease, yellow fever, Japanese

encephalitis and onchocerciasis, globally (World Heath Organization, 2016). Mosquitoes

are considered one of the most dangerous creatures on the planet because of their ability

to spread deadly diseases. In recent years the rate of infection has risen dramatically, and

a growing number of scientists are now concerned that global warming will translate into

an explosive growth of mosquito-borne diseases worldwide (Mosquito World, 2017).

Over 1 million people worldwide die from mosquito-borne diseases every year

(American Mosquito Control Association, 2014). Mosquito-borne diseases are those

spread by the bite of an infected mosquitoes. Not only can mosquitoes carry diseases that

afflict humans, they also transmit several diseases and parasites that dogs and horses are

very susceptible to. These include dog heartworm, West Nile virus (WNV) and Eastern

equine encephalitis (EEE). In addition, mosquito bites can cause severe skin irritation
 Larvicidal Effect of Used Coffee Grounds ...2

through an allergic reaction to the mosquito's saliva that causes the red bump and itching.

(American Mosquito Control Association, 2014). Diseases that are spread to people by

mosquitoes include Zika virus, Chikungunya virus, and dengue virus (Center for Disease

Control and Prevention, 2016).

Dengue fever is an endemic mosquito-borne disease in at least 100 countries in

Asia, the Pacific, the Americas, Africa, and the Caribbean (Center for Disease Control

and Prevention, 2014). According to Center for Disease Control and Prevention (CDC),

there are about 2.5 billion people or 40% of the world’s population are living in the areas

where there are risks of dengue transmission which is the most common occurring

mosquito borne disease. According to World Health Organization (WHO), there are

almost 50 to 100 million infections occurs annually, with 22,000 deaths mostly among

youngsters. In the 1970’s, it is one of the top causes of hospitalization and death among

children in the Philippines. Between 2004 and 2010, Philippines ranked seventh highest

number of dengue fever cases in the world. From January to June 2016, there have been

52,177 cases of dengue in the Philippines (InterHealth Worldwide, 2016).

Another virus that is also carried by mosquitoes is the Zika virus. Zika virus is an

emerging mosquito-borne virus that causes birth defects in babies whose mother is

infected with the disease. The newborn baby will have undeveloped head and brain

damage. It can be transmitted by a bite of a mosquito, sexually transmitted, through blood

transfusions and can be found in blood, semen, urine, saliva and the fluid in the eyes

(WebMD, 2005). In 2016, WHO declared Zika virus disease an emergency issue.

According to the review of Paixão, Barreto, Teixeira, Costa, and Rodrigues (2016), the
 Larvicidal Effect of Used Coffee Grounds ...3

overall attack rate of Zika illness reported in health care facilities was 14.6 per 1000

inhabitants where French Polynesia has the highest attack rate among patients presented

to health care facilities. Brazil also has an outbreak but is 3 to 20 times lower than French

Polynesia and Micronesia. There was an outbreak of Zika virus in the Philippines. In

November 2016, there are a total of 35 cases of Zika virus disease in the Philippines

(Aurelio, 2016). Localities most affected are the National Capital Region, Calabarzon,

Western Visayas, Central Luzon and Central Visayas (International Association of

Medical Assistance to Travellers (IAMAT), 2017).

Malaria is also one of the most common mosquito-borne disease in the world.

It is considered a life-threatening disease and transmitted through the bite of an

infected female Anopheles mosquito that carries the Plasmodium parasite (World Health

Organization, 2017). Between 2010 and 2015, malaria incidence among populations at

risk (the rate of new cases) fell by 21% globally. In that same period, malaria mortality

rates among populations at risk fell by 29% globally among all age groups, and by 35%

among children under 5. The WHO African Region carries a disproportionately high

share of the global malaria burden. In 2015, the region was home to 90% of malaria cases

and 92% of malaria deaths (World Health Organization, 2017).

The government has been finding ways to control the disease-carrying mosquitos

to decrease the increasing incidence of mosquito-borne diseases. In 2011, a team of

researchers from the Department of Science and Technology - Industrial Technology

Development Institute (DOST-ITDI) developed the Mosquito Ovicidal-Larvicidal (OL)

 Larvicidal Effect of Used Coffee Grounds ...4

trap system. It was launched in 2011 to address the increasing cases of dengue in the

Philippines. The said contraption can attract female Aedes mosquito thus making it an

easy prey for it to lay eggs that will no longer develop into adults. It has the mechanism

of stopping the proliferation of the deadly dengue carrying mosquitoes (Department of

Science and Technology, 2015).There have been studies that used coffee grounds can be

a potential larvicide. The studies of Itoyama and Bicudo (1992), Laranja, Manzatto and

Bicudo (2003), Lok (2003), Guirado and Bicudo (2007; 2015), Satho et al. (2015) have

demonstrated the larvidical activity of used coffee grounds.

Because of increasing cases of mosquito-borne diseases, we, the researchers have

chose this study to contribute to the ongoing public health problem worldwide and in the

locale through presenting a potential larvicide against mosquitoes. In our proposed study,

we aim to investigate the larvicidal activity of used coffee against mosquito larvae.
 Larvicidal Effect of Used Coffee Grounds ...5

Review of Related Literature

Mosquito-borne diseases

Mosquito-borne disease has the highest incident rate each year, about 1 million

people died of the said disease and hundreds of millions of people is experiencing and

suffering from the illness transmitted by mosquitoes (Oxitec, 2002). Mosquitoes are one

of the deadliest animals in the world which can carry and spread disease to the human

host (Urdahl, 2017).

The reactions of mosquito bites are associated with trauma produced by the

mechanical insertion of the proboscis by the mosquito. The initial cutaneous reaction is

due to sensitization effect of the saliva (Belizario Jr. & de Leon, 2004). The repeated

attacks themselves may be physically and psychologically damaging (McPherson &

Pincus, Henry's Clinical Diagnosis and Management by Laboratory Methods, 2011). A

lot of strains of virus and disease are carried by the mosquitoes, namely: dengue

hemorrhagic fever, malaria, Zika virus and chikungunya (Center for Disease Control and

Prevention, 2016). Usually, the female mosquitoes are the disease-carrying mosquito

because of the need of blood to nurture its babies and able to reproduce more larvae.

Because the Philippines is a tropical country, there were mosquitoes that can

multiply easily and a lot of people were being infected by many infectious diseases

carried by the mosquitoes (Department of Science and Technology, 2008).

 Larvicidal Effect of Used Coffee Grounds ...6

Dengue

Dengue fever is a viral disease and is transmitted by the bite female Aedes

aegypti and alborpictus. The mosquito lives very close to people and thrives in small

amounts of stagnant water. The Aedes aegypti is the primary vector in spreading the

disease; it usually bites during the day. Incubation of the disease is usually 4-10 days.

Symptoms include high fever for 1 to 4 days, severe headache, severe eye pain, joint

pain, muscle or bone pain, rashes, mild bleeding manifestation and low white blood cell

and platelet count (World Health Organization, 2015).

For the past 2 years the cases of dengue in the Philippines from January to June

increases. The case from 2015 with the said time frame was 36,972 and death toll of 129,

compared to 2016 which increases to 52,177 and a death toll of 207. There are

approximately 200,000 dengue cases in 2015 and 600 dengue related deaths. The worst

affected region was: Calabarzon (7,051 cases), Central Visayas (5,510 cases), Central

Luzon (5,452 cases), Northern Mindanao (5,044 cases) and SOCCSKSARGEN (4,075

cases). Dengue has become an all year-round disease which become endemic in the

country (InterHealth Worldwide, 2016).

Chikungunya

Chikungunya is a mosquito borne disease first discovered in 1952 at Tanzania. It

is an RNA virus that belong to the genus Alphavirus and family of Togaviridae.
 Larvicidal Effect of Used Coffee Grounds ...7

Chikungunya is derived from the Kinakonde language, means “to become contorted” or

“to walk bent over” (World Health Organization, 2016). It is transmitted by the infected

female mosquitos or via contact with infected blood. The disease can cause fever that

lasts for days, joint pains (polyarthralgia) which can last for days, weeks, months or even

year or more. When suspecting for chikungunya disease one must have these following

symptoms: fever (as high as 40 degrees), pain and swelling of the joints, headache, rashes

and muscle pains. Sometimes patient can have conjunctivitis or having nausea and

vomiting. The virus is rarely fatal but the patient can feel severity of the disease and

disability (Newman, 2016).

Cases of chikungunya have already rise higher than dengue virus. In 2011,

outbreak in Cagayan and Davao of the disease occurs when the tropical storm Sendong

hits. In 2012, 100 residents in Surigao was affected (ABS CBN News, 2012). In first half

of 2013, 157 positive cases have been reported. On July 25 of the same year, more than

300 cases were reported in San Nicolas, Ilocos Norte (Department of Health, 2013). In

2016, 470 suspected cases of the disease occur in Cavite (Bonalos, 2016).

Zika virus disease

Zika virus disease is a mosquito borne Flavivirus transmitted by the Aedes

mosquitoes, same mosquito that carries dengue, chikungunya and yellow fever. It was

first identified in Uganda in 1947. Zika virus disease causes birth defects in babies whose

mother is infected with the disease. The infected babies are born with undeveloped head

and brain damage. It can be transmitted sexually, bite of a mosquito, blood transfusion
 Larvicidal Effect of Used Coffee Grounds ...8

and can be found in blood, semen, urine, saliva and the fluid in the eyes (WebMD, 2005).

The most common symptom when you have Zika virus disease is rashes, fever, joint

pains, muscle pain and headache which can last for days to a week. To prevent infection

from Zika virus disease, avoid interacting with people who came from a country that has

a history of Zika virus disease, blood should be tested if it is infected by the virus and

prevent sexual transmission by using a condom or abstinence from sex (Center for

Disease Control and Prevention, 2017).

As of now there are 52 cases of Zika virus disease in the Philippines. 20 cases

came from National Capital Region, 18 cases from CALABARZON, 15 cases from

Western Visayas, 2 cases from Central Luzon and 2 cases from Central Visayas, leaving

Mindanao with 0 case (Hinayon, news.abs-cbn.com, 2017).

Mosquito

Mosquito, from the family of Culicidae of the order Diptera, are two winged

insects that have a long-scaled proboscis, usually covered with scales that vary the color

from white to almost black. They may also be group by their specific contrasting patterns

which will be referred to as white rings on all legs, stripes on the Scutum and bands on

the Abdomen. Their Antenna just forward from their heads are longer and more

numerous in males than in females. Only a female mosquito bite, male and female feed

mainly on fruit and plant nectar but the female also needs blood to help her eggs develop.

Mosquitoes are of major medically importance due to their role as vector for

transmission of various diseases. Mosquitoes from the subfamilies Anophelinae and

 Larvicidal Effect of Used Coffee Grounds ...9

Culicinae are public health important that provide a piercing-sucking mouth parts several

times longer than the head. The subfamily Toxorhynchitinae is represented by large and

brilliantly colored, non-bloodsucking mosquitoes whose larvae are predaceous on

animals smaller than them (Balatazar & Salazar, 1998).

There are some 110 genera and subgenera, and about 3,500 mosquitoes now at

present are belonging to the family Culicidae. There are 3 common and can spread a

human disease genus, the Anopheles (carry malaria), Culex (carry encephalitis and

filariasis) and Aedes (carry yellow fever, dengue and encephalitis).

Morphology

Figure 1. Mosquito: Aedes aegypti http://www.nbcnews.com/storyline/zika-virus-

outbreak/aedes-aegypti-meet-mosquito-spreading-zika-virus-panic-n504026

Mosquitoes are small (3-6 mm), two-winged insects belonging to the family

Culicidae of the order Diptera. They are easily distinguished from most other flies by a

combination of the following characters: a long proboscis projecting forwards from the

head; the presence of scales on the wing veins; a fringe of scales along the posterior
 Larvicidal Effect of Used Coffee Grounds ...10

margin of the wing; and a characteristic wing venation, the second, fourth and fifth

longitudinal veins being branched (Goma, 1966).

Males and females can be differentiated by the form of the antennae. In males they

are very plumose, while in females they only have a few short hairs. In most others

than Anopheles species the maxillary palps in the female are very short in contrast to the

male where they are longer than the proboscis. In both sexes of Anopheles, the maxillary

palps are long, but clubbed in the males.

They are Holometabola with the first stage differing completely from the last one

in form, structure and habits. Most of the common and important mosquitoes as

transmitter of pathogens for animal and man belong to four genera: Anopheles, Culex,

Mansonia and Aedes. (Companion Vector Borne Diseases, 2017).

Development

The yellow fever mosquito or the Aedes aegypti goes through a complete

metamorphosis with an egg, larvae, pupae and adult stage. The adult life span can range

from two weeks to a month depending on environmental conditions. The life cycle can be

completed one and a half to three weeks.

 Larvicidal Effect of Used Coffee Grounds ...11

Figure 2. Mosquito Life cycle http://www.wumcd.org/mosquito/lifecycle.gif

Figure 2 shows the life cycle of a mosquito. After taking a blood meal, female

Aedes aegypti produce on average 100 to 200 eggs per batch. The eggs are smooth, long,

ovoid shaped, and roughly one millimeter long. When first laid, eggs appear white but

within minutes turn a shiny black (Uniyal, et al., 2016). In warm climates, eggs may

develop in just two days, whereas in cooler temperate climates, development can take up

to a week (Arun, 2010). Laid eggs can survive for very long periods in a dry state, often

for more than a year. However, they hatch immediately once submerged in water. This

makes the control of the dengue virus mosquito very difficult. Most of the larval stage is

spent at the water’s surface.

Larvae emerge from mosquito eggs, but only after the water level rises to cover

the eggs. This means that rainwater or humans adding water to containers with eggs will

trigger the larvae to emerge (Center for Disease Control and Prevention, 2012). The

larval stage is completed within 4 to 9 days. The larvae pass through four instars,

spending a short amount of time in the first three and up to three days in the fourth instar.
 Larvicidal Effect of Used Coffee Grounds ...12

Fourth instar larvae are approximately eight millimeters long. Larval development is

temperature dependent. If temperatures are cool, Aedes aegypti can remain in the larval

stage for months so long as the water supply is sufficient (Uniyal, et al., 2016).

After the fourth instar, larvae enter the pupal stage. The pupal stage lasts for 1 to 5

days. Pupae will develop until the body of the newly formed adult flying mosquito

emerges from the pupal skin and leaves the water (Uniyal, et al., 2016).

Adult: Male and female adults feed on nectar of plants; however, female

mosquitoes need blood to produce eggs, and are active in the daytime (Center for Disease

Control and Prevention, 2012).

Habitat

Mosquitoes prefer stagnant water within which to lay their eggs. They most

commonly infest ponds, marshes, swamps and other wetland habitats. However, they can

thrive in a variety of locations and can successfully grow in numbers even when not in

their natural habitat. Many species of mosquitoes use containers of water as egg-deposit

sites. Aedes aegypti utilizes both natural and artificial habitats. Their habitats may also be

either terrestrial or aquatic.

Naturally they utilized tree holes and plant axils (leaf joints) found in forested

areas as aquatic habitats. And artificially at artificial containers frequently associated with

human habitation which include vases, water tanks and tires. They also use aquatic

habitats such as septic tanks which are located underground and capable of using aquatic
 Larvicidal Effect of Used Coffee Grounds ...13

habitats. Aedes aegypti larvae require standing water for the completion of their growth

cycle.

Therefore, potential breeding habitats are places that contain standing bodies of

water. They also prefer dark colored containers located in the shade. Aedes aegypti is

widely distributed through the tropical and subtropical areas of the world (Dom, Hassan,

& Ahmad, 2013).Hot, humid environments are most amenable to mosquito growth and

survival. Infestations can occur easily in tropical areas. Some species have also been

known to inhabit freezing locations such as the Arctic Circle (Orkin, 2017).

Feeding Habits

Female mosquitoes bite because they need protein found in blood to develop their

eggs. They choose their targets through a combination of smell, heat and visual cues, and

continue seeking bloodmeals until their abdomens are full. The females can live up to

about a month and feed every two or three nights during that time. The most common

mosquito species feed at dawn and dusk, and for a few hours into dark. However, there

are some species that are particularly aggressive and will feed both day and night.

Mosquitoes locate bloodmeals first by using sensors on their antennae to detect trace

amounts of carbon dioxide released in human breath. That CO2 rises in plumes that

mosquitoes can sense from hundreds of feet away (Mosquito World, 2017).
 Larvicidal Effect of Used Coffee Grounds ...14

The female Aedes aegypti feeds almost exclusively on human blood to produce

eggs, and are active in the daytime (Center for Disease Control and Prevention, 2012).

They feed on a lower frequency of other hosts including bovine, swine, cat, rat, and

chicken which represents less than 1% of blood meals (Ponlawat & Harrington, 2005).

The males do not attempt to puncture skin. Organic particulate matter present in the water

provide food for larvae. Like, small aquatic organisms, algae and particles of plant and

animal material in water-filled containers (Zettel & Kaufman, 2013).

Mosquito Ovicidal-Larvicidal (OL) Trap

Figure 3. Mosquito Ovicidal-Larvicidal Trap.

(DOST Mosquito Ovicidal/Larvicidal (OL) Trap for Dengue Prevention, 2013)

Larvicide is an agent killing for larval pests (Merriam-Webster, 2017). The

problem caused by the mosquito species, Aedes aegypti, is of importance since it killed
 Larvicidal Effect of Used Coffee Grounds ...15

millions every year. Therefore, scientists extended their capacity to help in eradicating or

lowering the rate of spread of these mosquito-borne diseases.

In our locale, the S&T Media Service (2015) made an article about a team of

researchers who developed an organic method to reduce the rate of mosquitoes and to a

success was awarded for developing the Mosquito Ovicidal-Larvicidal (OL) trap system

during the 114th Philippine Civil Service Anniversary in Malacañang Palace, Manila.

The OL trap was launched in 2011 to address the burgeoning problem of dengue

infection. The DOST-ITDI OL trap research team designed the trap to detect, monitor,

and control the population of the dengue-carrying Aedes aegypti mosquitoes.

The simple contraption consists of an ordinary can (the size of a regular evap.

milk) painted board (1x6.5x1/2 in.). Latest development replaced the can with black

plastic cans of the same size. The solution (pellets are dissolved in tap water) is

introduced into this can and by capillary action moistens the board, making it ideal for the

female Aedes mosquito (FAM), the dengue carrying mosquito to lay its eggs on. Black

normally attracts FAM thus making the system an easy prey for it to lay eggs that will no

longer develop into adults, therefore stopping proliferation of the deadly dengue carrying

mosquitoes (Department of Science and Technology, 2015).

In a nationwide mosquito-reduction project, the DOST, from June to December

2012, produced one million OL trap kits and distributed these to all public elementary

and high schools nationwide. A website was launched in 2013, it is designed to alert

dengue coordinators, city health officers, and other health practitioners on the mosquito
 Larvicidal Effect of Used Coffee Grounds ...16

density and the possibility of dengue transmission in their respective areas (S&T Media

Service, 2015).

Coffee

Coffee has been for decades the most commercialized food product and most

widely consumed beverage in the world. It is considered a functional food, primarily due

to its high content of compounds that exert antioxidant and other beneficial biological

properties (Chu, 2012).

Properties

Coffee, they are stronger, more resistant to pests and disease, and less demanding

than other types of coffee. It prefers higher temperatures: 24-30°C (75-86° F), and more

rainfall: 2000–3000 millimeters (79-118 inches). And grows in lower elevations, from

sea level to 700 meters (about 2,300 feet), as well as 40-50 % higher caffeine levels.

Caffeine

Figure 4. Chemical Structure of Caffeine

(California State University, 2009)
 Larvicidal Effect of Used Coffee Grounds ...17

Caffiene is classified as a central nervous stimulant and an ergogenic compound.

The active ingredient in coffee is 1,3,7- trimethylxanthine, and is metabolized primarily

in the liver, with assistance from the kidneys and brain. Worldwide, the average daily

consumption of caffeine is 70 mg, or the equivalent of one small cup of round coffee per

person besides coffee, caffeine is also present in chocolate, kola nut, soft drinks,

caffeinated teas, and various prescription and over-the-counter drugs (Aaronshelley,

2016). Caffeine is a natural component of coffee, tea, guarana and chocolate. It stimulates

the central nervous system as an adenosine-receptor antagonist. Caffeine metabolites,

especially 1-methylxantine and 1-methylurate, have exhibited antioxidant activity in

vitro, and the in vivo iron-reducing capacity of regular coffee is higher than that of

decaffeinated coffee (Farah, 2012).

Effects of Caffeine to Mosquitoes

Caffeine have revealed to have an effect to mosquitoes. In the study conducted by

Laranja, Manzatto and Bicudo (2003), caffeine and used coffee grounds blocks the

development of Aedes aegypti in its early stages. In their study, 300 mg/ml concentration

has the most efficient effect, producing 100% of larval mortality until nine days after

preparation. Significant results were observed in terms mortality rate, longevity and

esterase patterns using caffeine and used coffee grounds compared to other treatments

used. A study was also conducted by Lok (2003) about the effect of caffeine to

mosquitoes which aimed to know if caffeine could be a future larvicide and to find if

caffeine is harmful especially to human beings. Results showed that caffeine would
 Larvicidal Effect of Used Coffee Grounds ...18

influence mosquitoes and can be a future larvicide for mosquito control. In the study of

Guirado and Bicudo (2007) about the effects of used coffee grounds on larval mortality

of Aedes aegypti by suspension concentration and age versus efficacy, they evaluated the

duration of the effect on larval mortality of aqueous suspensions of used coffee ground in

different concentration and the results obtained was significant and it strengthens the

validity of considering used coffee ground preparations as possible alternative control of

Aedes aegypti. Another study brought up by Guirado and Bicudo (2015) is about the

attractiveness of bio insecticides caffeine and used coffee grounds in the choice of

oviposition site by Aedes aegypti. The results showed that there is potential of using used

coffee grounds as auxiliaries in the control of Aedes aegypti.

Theoretical Framework

Previous studies have been discovered that the used of coffee grounds help to

reduce the rapid growth population of mosquito particularly Aedes aegypti. Coffee

contains large amounts of chemicals many of which are high toxic to the developing

larvae of mosquito which has been hypothesized by Satho et al. (2015). Coffee also

decreases the reproductive capacity of mosquitoes which helps to lessen the laying of

eggs for female mosquitoes (Laranja, A., Manzatto, A. and Bicudo, H., 2003). The use of

caffeine as an insect repellent may results in distorted behavior by suppressing enzymes

in the insects’ nervous systems (Nehlig, Daval, & Debry, 1992). With the following

literatures, it was suggested that caffeine can be used for alternative insect control. The

afore- mentioned studies played an important role in developing our research study
 Larvicidal Effect of Used Coffee Grounds ...19

Conceptual Framework

Independent Variable Dependent Variable

Coffee ground concentrations:

- 0.5 g

- 1g

- 1.5g
Mortality rate of the mosquito
- 2g

- 2.5 g

- 3g

Positive control

- Mosquito OL trap

Negative control

- Distilled water

Figure 1. Conceptual Framework showing the relationship of the independent and

dependent variable.

The prepared coffee ground will be extracted with the following mass of 0.5g, 1.0g, 1.5g,

2.0g, 2.5g, and 3.0g. Mosquito OL trap will act as a positive control and distilled water

will act as a negative control. The concentration of coffee ground was manipulated to

cause changes in the dependent variable, which is the mortality of the larvae of mosquito

in the study.
 Larvicidal Effect of Used Coffee Grounds ...20

Statement of the Problem

This study aims to investigate the larvicidal effect of used coffee grounds against

Aedes aegypti and specifically answers the following:

1. Is coffee grounds effective for mosquito?

2. What is the mortality rate of mosquito larvae when expose to different concentrations

of coffee grounds?

2.1. 0.5 g;

2.2. 1.0 g;

2.3. 1.5 g;

2.4. 2.0 g;

2.5. 2.5 g; and

2.6. 3.0 g?

3. What is the mean mortality rate of Aedes aegypti larvae at 5 days incubation in

Mosquito OL trap?

4. Is there a significant difference in the mean mortality rate of the different

concentrations of coffee and the Mosquito OL trap?

Null Hypothesis

𝐻𝑜1 : There is no significant difference in the mean mortality rate of mosquito larvae

when exposed to different volumes of the coffee grounds and in Mosquito OL trap.
 Larvicidal Effect of Used Coffee Grounds ...21

CHAPTER II

METHODS

This chapter discusses the design, setting, procedures of the study and what is

applicable for this study. It specifies the methods of research to be used, research design,

data gathering, instruments to be used, and statistical analysis of gathered data.

Design

The design of this study will be experimental posttest with control design. It is a

type of experimental design where test units are randomly allocated to an experimental

group and a control group. This design uses two groups, one group is given the treatment

and the results are gathered at the end. The control group receives no treatment, over the

same period, but undergoes the same tests.

In our study, the coffee concentrations will be manipulated while the positive and

negative control will be the Mosquito OL Trap and distilled water, respectively. The

researchers will test the mortality rate of mosquito larvae exposed in the different

treatments after five (5) days of incubation.

 Larvicidal Effect of Used Coffee Grounds ...22

Subjects

Used coffee grounds that will be used in this study will be ordered from selected

coffee shops in Davao City. Mosquitoes will be isolated in Sarphil Village, Agdao,

Davao City. It is a residential area with canals and stagnant bodies of water that is a

potential breeding place of mosquitoes. In 2016, Agdao is one of the many identified

hotspots in Davao City for dengue fever (SunStar Davao, 2016). Mosquito larvae pass

through four instars, wherein they presented growth periods with highly similar

durations. In this study, the researchers ten (10) larvae per treatment.

Mosquito OL Trap will be requested from the Department of Science and

Technology (DOST). The simple contraption consists of black plastic cans and a solution

is made up of pellets that are dissolved in tap water. Two (2) packs of pellets is the

standard usage of the said trap.

Measures

The mortality rate of mosquito larvae in different concentrations will be measured

by separation according to their sizes and morphology. The larval stage and the pupal

stage are different. Larvae are about 1-1.5 millimeters long when newly hatched and

grow to a full length of eight (8) millimeters (Belizario Jr. & de Leon, Philippine

Textbook of Medical Parasitology, 2004). Pupae are mobile and the non-feeding stage.

The abdomen is narrow, segmented and curved towards the ventral side (Medhekar,

2016). The larvae and pupae will be carefully separated using a very fine mesh to filter.

The larvae that have not developed into pupae or into adult will be recorded.
 Larvicidal Effect of Used Coffee Grounds ...23

Research Procedure

Approval from the Ethics Committee

Transmittal Letters will be sent to the Department Dean and Director of Laboratories

Implementation of the Experiment

Isolation and Speciation of Mosquitoes

Breeding of Mosquitoes

Collection of Used Coffee Grounds

Preparation of Treatments and Controls. 0.5 g/ml, 1.0 g/ml, 1.5 g/ml, 2.0 g/ml, 2.5
g/ml, 3.0 g/ml for coffee concentrations, Mosquito OL Trap (4 packs of pellets
dissolved in 250 ml water) and distilled water for positive and negative control

Larvae will be transferred accordingly in each cups that contained different volumes of
used coffee grounds, positive, and negative control.

Data will be collected after five (5) days of incubation

Experiment will be carried out for three (3) trials to ensure certainty of results.

Figure 6. Procedure in Mosquito Larvae Collection and Treatment Application

 Larvicidal Effect of Used Coffee Grounds ...24

Data Analysis

To compute for the percentage of mean mortality rate, Cohort Survival Rate will

be used, in which we will determine the percentage of mortality rate of the treated larvae.

It is a study to determine the correlation of the absolute risk of subject contraction.

Specifically, we will use the standardized mortality ratio, and can be calculated by

dividing the observed number of dead cases (larvae) in the cohort by the expected

number of deaths (Ressing, 2010).

Limitations of the Study

Although the research will be carefully prepared, its design is not without flaws

and there were still some unavoidable limitations.

First, the methods for trapping the Aedes aegypti mosquitoes is not yet fully

established since the researchers have not yet undergone in the seminar that is being

offered by the Department of Health. Once the seminar will be pushed through, the

researchers will have enough knowledge for the latter issue.

Second, because of the limited time given and lack of availability of subjects to be

used, this research will be conducted only on a small number of Aedes aegypti larvae,

specifically 10 per treatment (there are 8 treatments in total).

Lastly, because of the threat of acquiring mosquito borne diseases, we are

confined to do our experiment in a closed controlled environment in our school where

there are small possibilities to spread disease.

 Larvicidal Effect of Used Coffee Grounds ...25

Ethical Considerations

Many factors are needed to be considered when dealing biological organisms

because it is not only hazardous but also takes time.

Wearing of proper personal equipment is a must. When dealing with mosquitoes,

your skin needs to be fully covered to avoid the possibility of acquiring mosquito-borne

diseases.

Proper disposal of chemicals should be observed to avoid spreading harm to

another incognizant individual.

The individuals performing the extraction should be knowledgeable of the proper

handling of specimen to avoid unnecessary accidents. It should be noted when lives of

any organism, in our case, the lives of the mosquito should be considered. The

mosquitoes should be handled in an enclosed area where they could not escape and may

potentially spread an endemic.

 Larvicidal Effect of Used Coffee Grounds ...26

REFERENCES

Aaronshelley. (2016, December 11). Properties and Characteristics of Caffeine. Retrieved from
Aaronshelley: http://aaronshelley.com/properties-and-characteristics-of-caffeine/

Abrantes, Y., Andan, R., Arellano, K., Artes, K., Balmorez, J., Castillones, M., . . . Tamondong, T.
(2012). The Efficacy of the Ovicidal/Larvicidal (OL) Trap Pellets on the Mortality of
Mosquito Larvae. Davao City, Philippines.

ABS CBN News. (2012, September 26). Chikungunya' affects 100 residents in Surigao. Retrieved
march 31, 2017, from ABSCBN News: http://news.abs-
cbn.com/nation/regions/09/26/12/chikungunya-affects-100-residents-surigao

al., T. e. (2015). Caffeine impairs resection during DNA break repair by reducing the levels of
nucleases Sae2 and Dna2. Retrieved from National Center for Biotechnology
Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538808/

American Mosquito Control Association. (2014). Mosquito-Borne Diseases. Retrieved from

American Mosquito Control Association: www.mosquito.org/mosquito-borne-diseases

Arun, N. (2010, August 31). Tips for prevention of breeding of Aedes mosquito in your urban
neighbourhood. Retrieved from A Malayi Doctor's Blog:
http://malayalidoc.blogspot.com/2010/08/tips-for-prevention-of-breeding-of.html

Aurelio, J. (2016). 2 new Zika victims bring total number of cases in PH to 35. Manila: Philippine
Daily Inquirer.

Balatazar, C., & Salazar, P. (1998). Aedes aegypti. Retrieved from ITIS Report:
https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=126
240#null

Belizario Jr., V., & de Leon, W. (2004). Philippine Textbook of Medical Parasitology (2nd ed.).
Manila: University of the Philippines - Manila.

Belizario Jr., V., & de Leon, W. (2004). Philippine Textbook of Medical Parasitology. Manila:
University of the Philippines - Manila.

Bhawani, S. F. (2015). Spectrophotometric Analysis of Caffeine. International Journal of

Analytical Chemistry, 2015, 7. doi:10.1155/2015/170239

Bonalos, P. (2016, October 4). Cavite town under State of Calamity due to chikungunya.
Retrieved april 1, 2017, from cnnphilippines.com:
http://cnnphilippines.com/regional/2016/10/03/cavite-town-under-state-of-calamity-
due-to-chikungunya.html

Bota, S., Mariana, G., Corina, M., & Caraban, A. (2015). Method For Quantitative Determination
Caffeine From Coffee. Retrieved from portmed:
 Larvicidal Effect of Used Coffee Grounds ...27

http://protmed.uoradea.ro/facultate/publicatii/ecotox_zooteh_ind_alim/2015B/imapa/
05._Bota_Sanda.pdf

Brenner, M., & Thoms, S. (1984). Caffeine blocks activation of cyclic AMP synthesis in
Dictyostelium discoideum. Retrieved from National Center for Biotechnology
Information: https://www.ncbi.nlm.nih.gov/pubmed/6319207

Brittanica. (2009). Spectrophotometry. Retrieved April 25, 2017, from Brittanica.com:

https://www.britannica.com/science/spectrophotometry

California State University. (2009). High Performance Liquid Chromatography (Chemistry 321L
Manual). Retrieved April 24, 2017, from csun.edu:
http://www.csun.edu/~hcchm003/321l/321lmlc.pdf

Center for Disease Control and Prevention. (2010). Mosquito borne diseases. Retrieved from
Center for Disease Control and Prevention: cdc.gov

Center for Disease Control and Prevention. (2012). Aedes aegypti Life Cycle. Retrieved from
Center for Disease Control and Prevention:
https://www.cdc.gov/dengue/resources/factSheets/MosquitoLifecycleFINAL.pdf

Center for Disease Control and Prevention. (2015, August 5). Japanese Encephalitis. Retrieved
march 25, 2017, from Center for Disease Control and Prevention:
http://www.cdc.gov/japaneseencephalitis/symptoms/index.html

Center for Disease Control and Prevention. (2016, November 8). Mosquito borne diseases.
Retrieved march 24, 2017, from Center for Disease Control and Prevention:
http://www.cdc.gov/niosh/topics/outdoor/mosquito-borne/

Center for Disease Control and Prevention. (2017, March 9). Zika: Overview. Retrieved march 26,
2017, from Center for Disease Control and Prevention:
http://www.cdc.gov/zika/about/overview.html.

Chem LibreTexts. (2017). High Performance Liquid Chromatography. Retrieved May 13, 2017,
from chem.libretexts.org:
https://chem.libretexts.org/Core/Analytical_Chemistry/Instrumental_Analysis/Chromat
ography/High_performance_liquid_chromatography

Chemeketa Commmunity College. (2000). Extraction of Caffeine from Tea. Retrieved May 14,
2017, from Chemeketa.edu: http://faculty.chemeketa.edu/jcammack/CH241-
3B%20Lab/CH241B%20Labs/CH241%206%20Caffeine%20Extraction%20F14.pdf

Chemistry LibreTexts. (2015). Spectrophotometry. Retrieved April 30, 2017, from

chem.libretexts.org:
https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kinetics/Reactio
n_Rates/Experimental_Determination_of_Kinetcs/Spectrophotometry
 Larvicidal Effect of Used Coffee Grounds ...28

Companion Vector Borne Diseases. (2017). Mosquito Borne Diseases. Retrieved from Companion
Vector Borne Diseases.

Department of Health. (2013). Retrieved march 31, 2017, from Department of Health:
www.doh.gov.ph/node/1494

Department of Science and Technology. (2008). The mosquito menace in the Philippines.
Retrieved march 24, 2017, from Philippine Council for Health Research and
Development : http:/www.pchrd.dost.gov.ph/index.php/news/library-health-
news/5689-the-mosquito-menance-in-the-philippines

Department of Science and Technology. (2015). DOST Mosquito Trap Researchers bag Dangal ng
Bayan Presidential Award. Retrieved from Department of Science and Technology:
dost.gov.ph/knowledge-resources/news/44-2015-news/647-dost-mosquito-trap-
researchers-bag-dangal-ng-bayan-presidential-award

Department of Science and Technology. (2015). OL Trap. Retrieved from Industrial Technology
Development Institute: http://www.itdi.dost.gov.ph/index.php/21-technology-
features/120-mosquito-ol-trap

Dom, N., Hassan, A., & Ahmad, I. (2013, March). Habitat characterization of Aedes species
breeding in urban hotspot areas. Retrieved from ScienceDirect:
http://www.sciencedirect.com/science/article/pii/S1877042813024695

DOST Mosquito Ovicidal/Larvicidal (OL) Trap for Dengue Prevention. (2013). Retrieved June 1,
2017, from Filipino Inventions and Discoveries:
http://filipinoinventionsanddiscoveries.blogspot.com/2013/07/dost-mosquito-
ovicidallarvicidal-ol_4.html

Farah, A. (2012). Coffee: Emerging Health Effects and Disease Prevention.

Farrah, A. (2012). Coffee Constituents. Retrieved from Institute of Food Technologists:

http://www.ift.org/~/media/Knowledge%20Center/Publications/Books/Samples/IFTPre
ssBook_Coffee_PreviewChapter.pdf

Ferguson, I. (2013). What are the Differences Between Arabica and Robusta Coffee Beans?
Retrieved May 20, 2017, from coffeebuyer.co.uk:
http://www.coffeebeansblog.co.uk/what-are-the-differences-between-arabica-and-
robusta-coffee-beans/

Gouge, D., Shujuan, L. W., Summer, C., Nair, S., & Olson, C. (2016, June). Mosquitoes: Biology
and Integrated Mosquito Management. Retrieved from University of Arizona College of
Agriculture and Life Sciences:
https://extension.arizona.edu/sites/extension.arizona.edu/files/pubs/az1706-2016.pdf
 Larvicidal Effect of Used Coffee Grounds ...29

Guevarra, B. Q., Aguinaldo, A. M., Castro-Bernas, G. d., Claustra, A. L., Espeso, E. I., Gonzales, R.
E., . . . Ysrael, M. C. (2005). A Guidebook to Plant Screening: Phytochemical and
Biological. Espana, Manila: UST Publishing House .

Guirado, M., & Bicudo, H. (2007). Effect of Used Coffee Grounds on Larval Mortality of Aedes
aegypti L. (Diptera: Culicidae): Suspension Concentration and Age versus Efficacy.
Retrieved from Bioassay : https://bioassay.org.br/bioassay/article/view/52/84

Guirado, M., & Bicudo, H. (2015). Attractiveness of bioinsecticides caffeine and used coffee
grounds in the choice of oviposition site by Aedes aegypti (Diptera: Culicidae).

Healthline. (2017). Malaria. Retrieved from Healthline:

http://www.healthline.com/health/malaria#prevention8

Herriman, R. (2015, March 22). Japanese encephalitis in the Philippines: Vaccine should be part
of national schedule according to researchers. Retrieved from Outbreak News Today:
http://outbreaknewstoday.com/japanese-encephalitis-in-the-philippines-vaccine-
should-be-part-of-national-schedule-according-to-researchers-95131/

Hinayon, R. (2017, february 3). Retrieved april 1, 2017, from news.abs-cbn.com: news.abs-
cbn.com/news/

Hinayon, R. (2017). 57 Zika cases in PH; DOH verifying microcephaly case. Retrieved April 1,
2017, from ABS CBN News: http://news.abs-cbn.com/news/02/03/17/57-zika-cases-in-
ph-doh-verifying-microcephaly-case

Imam, H., Zarnigar, Sofi, G., & Seikh, A. (2014). The basic rules and methods of mosquito rearing
(Aedes aegypti). Retrieved April 30, 2017, from National Center for Biotechnology
Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992806/

InterHealth Worldwide. (2016, July 07). Dengue fever in the Philippines. Retrieved march 31,
2017, from InterHealth Worldwide:
http://www.interhealthworldwide.org/home/health-resources/health-
alerts/2016/july/06/denge-fever-rise-in-the-philippines/

International Association of Medical Assistance to Travellers (IAMAT). (2017, February 28).

General Health Risks: Zika Virus. Retrieved from International Association of Medical
Assistance to Travellers: https://www.iamat.org/country/philippines/risk/zika-virus

Kamaraj, C., Bagavan, A., Elango, G., Abduz Zahir, A., Rajakumar, G., Marimuthu, S., . . . Abdul
Rahuman, A. (2011, July). Larvicidal activity of medicinal plant extracts against
Anopheles subpictus & Culex tritaeniorhynchus. Retrieved from National Center for
Biotechnology Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171902/

Kritz, B. (2017, February). Japanese encephalitis on the rise in PH. Retrieved april 1, 2017, from
The Manila Times: www.manilatimes.net/japanese-encephalitis-rise-ph/314588
 Larvicidal Effect of Used Coffee Grounds ...30

Laranja, A., Manzato, A., & Bicudo, C. (2003). Effects of Caffeine and used coffee grounds on
biological features of Aedes aegypti (Diptera: Culicidae) and thei possible use in
alternative control. Retrieved from National Center for Biotechnology Information:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436121/#CR63

Laranja, A., Manzatto, A. and Bicudo, H. (2003). Effects of caffeine and used coffee grounds on
biological features of Aedes aegypti (Diptera, Culicidae) and their possible use in
alternative control. Retrieved from
http://scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572003000400004

Lok, D. (2003). The Effect of Caffeine to Mosquitoes. Retrieved from California State Science Fair:
cssf.usc.edu/History/2003/Projects/J1421.pdf

Lokker, B. (2014). Introduction to Coffee Plant. Retrieved from

http://www.coffeecrossroads.com.

Lozano-Fuentes, S., Hayden, M., Carlos, W.-R., Ochoa-Martinez, C., Tapia-Santos, B., Kobylinski,
K., . . . Eisen, L. (2012, November 7). The Dengue Virus Mosquito Vector Aedes aegypti at
High Elevation in México. Retrieved from The American Journal of Tropical Medicine and
Hygiene: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516267/#R1

Maynard, A. (2016, February 3). Three ways synthetic biology could annihilate Zika and other
mosquito-borne diseases. Retrieved from The Conversation:
http://theconversation.com/three-ways-synthetic-biology-could-annihilate-zika-and-
other-mosquito-borne-diseases-54087

McPherson, R., & Pincus, M. (2011). Henry's Clinical Diagnosis and Management by Laboratory
Methods. Elsevier.

McPherson, R., & Pincus, M. (2011). Henry's Clinical Diagnosis and Management by Laboratory
Methods (22nd ed.). Elsevier.

Medhekar, A. (2016). Structure and Life Cycle of Mosquito (With Diagram). Retrieved May 7,
2017, from Biology Discussion: http://www.biologydiscussion.com/articles/structure-
and-life-cycle-of-mosquito-with-diagram/2675

Morgan, S., Koren, G., & Bozzo, P. (2013). Is caffeine consumption safe during pregnancy?
Retrieved from National Center for Biotechnology Information:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625078/

Mosquito World. (2017). Mosquito World. Retrieved from Mosquito Diseases:

http://www.mosquitoworld.net/when-mosquitoes-bite/diseases

National Health Service Choice. (2016, February 25). Japanese Encephalitis. Retrieved march 25,
2017, from nhs.uk: www.nhs.uk/conditions/japan-encephalitis/pages/introduction.aspx
 Larvicidal Effect of Used Coffee Grounds ...31

Nehlig, A., Daval, J., & Debry, G. (1992). Caffeine and the centralnervous system: mechanisms of
action, biocehmical, metabolic and psychostimulant effects. Retrieved from National
Center for Biotechnology Information: https://www.ncbi.nlm.nih.gov/pubmed/1356551

Newman, T. (2016, February 24). chikungunya: Cause,Diagnosis and Treatment. Retrieved march
24, 2017, from medicalnewstoday: www.medicalnewstoday.com/artcles/306328.php

Oestreich-Janzen, S. (2010). Nitrogenous Compounds I: Caffeine. In Chemistry of Coffee (Vol. 3,

p. 9). Hamburg, Germany: Elsevier. Retrieved April 28, 2017, from
https://booksite.elsevier.com/brochures/conap2/PDFs/Vol3_Chemistry_of_Coffee_lr.pd
f

Orkin. (2017). Mosquito Habitats. Retrieved from Orkin:

https://www.orkin.com/other/mosquitoes/mosquito-habitats/

Oxitec. (2002). Mosquito borne disease. Retrieved march 24, 2017, from Oxitec:
www.oxitec.com/mosquito-borne-disease/

Paixão, E., Barreto, F., Teixeira, M., Costa, M., & Rodrigues, L. (2016, April). History,
Epidemiology, and Clinical Manifestations of Zika: A Systematic Review. Retrieved from
American Journal of Public Health:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816002/

Ponlawat, A., & Harrington, L. (2005, September). Blood feeding patterns of Aedes aegypti and
Aedes albopictus in Thailand. Retrieved from National Center for Biotechnology
Information: https://www.ncbi.nlm.nih.gov/pubmed/16363170

Ressing, M. B. (2010, March 19). Data Analysis of Epideiological Studies. Retrieved April 7th,
2017, from Dtsch Arttebl Int.:
www.ncbi.nlm.nih.gov/pmc/articles/PMC2853157/#__ffn_sectitle

S&T Media Service. (2015, January 26). DOST Mosquito Trap Researchers Bag Dangal Ng Bayan
Presidential Award. Retrieved from Department of Science and Technology:
http://www.dost.gov.ph/knowledge-resources/news/44-2015-news/647-dost-
mosquito-trap-researchers-bag-dangal-ng-bayan-presidential-award

S. Oestreich-Janzen. (2010). Chemistry of Coffee. Vol 3.

Satho, T., Dieng, H., Ahman, M., Ellias, S., Hassan, A., Abang, F., . . . and Nolasco-Hipolito, C.
(2015). Coffee and its waste repel gravid Aedes albopictus females and inhibit the
development of their embryos. Retrieved from National Center for Biotechnology
Information: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436121/

Saxena, R., Harshan, V., Saxena, A., Sukumaran, P., M., S., & Kumar, M. (1993, March). Larvicidal
and chemosterilant activity of Annona squamosa alkaloids against Anopheles stephensi.
Retrieved from Journal of the American Mosquito Control Association:
https://www.ncbi.nlm.nih.gov/pubmed/8468579
 Larvicidal Effect of Used Coffee Grounds ...32

Sturelid, S. (1976). Enhancement by caffeine of cell killing and chromosome damage in Chinese
hamster cells treated with thiotepa. Retrieved from Wiley Online Library:
http://onlinelibrary.wiley.com/doi/10.1111/j.1601-5223.1977.tb01393.x/pdf

SunStar Davao. (2016, July 18). Dengue cases rising in Davao Region. SunStar. Retrieved May 8,
2017, from http://www.sunstar.com.ph/davao/local-news/2016/07/18/dengue-cases-
rising-davao-region-486137

Truttman, J. (2002, July). High Performance Liquid Chromatography. Retrieved April 24, 2017,
from Pomona.edu:
https://pages.pomona.edu/~wsteinmetz/chem160/HPLC_labmanual.doc

United States Environmental Protection Agency. (2016, December 19). Permethrin, Resmethrin,
d-Phenothrin (Sumithrin®): Synthetic Pyrethroids For Mosquito Control. Retrieved from
US EPA: https://www.epa.gov/mosquitocontrol/permethrin-resmethrin-d-phenothrin-
sumithrin

Uniyal, A., Tikar, S., Singh, R., Shakti, V., Shukla, O., Agrawal, P., . . . Sukumaran, D. (2016, June).
Repellent effect, knockdown study and electrophysiological responses of essential oils
against Aedes aegypti. Retrieved from Shodhganga @ INFLIBNET:
http://shodhganga.inflibnet.ac.in/bitstream/10603/70517/8/6%20material%20and%20
methods.pdf

Urdahl, K. (2017, March 8). Mosquitoes - one of the deadliest animals in the world. Retrieved
from GARD: http://www.gard.no/web/updates/content/22915020/mosquitoes-one-of-
the-deadliest-animals-in-the-world

WebMD. (2005). Retrieved march 25, 2017, from http://www.webmd.com/a-to-z-guides/zika-

virus-symptoms-prevention

WebMD. (2017). Retrieved march 25, 2017, from webmd.com: www.medmd.com/a-to-z

guides/zika-virus-symptom-prevention

Welder, D. C. (2017). Rotary Evaporator (rotavap). Dartmouth College, Hanover, New

Hampshire, United States of America.

World Health Organization. (2015). Mosquito borne diseases. Retrieved from World Health
Organization: who.int

World Health Organization. (2015). Mosquito borne diseases. Retrieved march 24, 2017, from
World Health Organization:
http://www.who.int/neglected_disease/vector_ecology/mosquito-borne-disease/en/

World Health Organization. (2016). Chikungunya. Retrieved march 24, 2017, from World Health
Organization: www.who.int/mediacentre/factsheets/fs327/en/

World Health Organization. (2017, April). Malaria. Retrieved from World Health Organization:
http://www.who.int/mediacentre/factsheets/fs094/en/
 Larvicidal Effect of Used Coffee Grounds ...33

World Health Organozation. (2015, December). Japanese encephalitis. Retrieved march 25,
2017, from World Health Organozation:
http://www.who.int/mediacentre/factsheets/fs386/en/

World Heath Organization. (2016, February). Vector-borne diseases. Retrieved from World
Health Organization: www.who.int/mediacentre/factsheets

Zettel, C., & Kaufman, P. (2013). Yellow fever mosquito Aedes aegypti (Linnaeus) (Insecta:
Diptera: Culicidae). Retrieved from University of Florida IFAS Extension:
http://edis.ifas.ufl.edu/in792