Bamboo (Bambusa vulgaris) as Larvicidal against

Mosquito (Aedes aegypti) Larvae

Chapter I

Introduction

1.1 Background of the Study

Mosquitoes are believe to be one of the dangerous insects in the world, as they bring

diseases that is fatal to mankind. In the Philippines, mosquito borne illnesses such as malaria,

dengue, filariasis and chikungunya remain to be a major public health concern. Dengue fever is a

mosquito-borne viral infection wherein children are the ones usually affected. The Department of

Health has documented around 30,000 cases of dengue all over the Philippines from January to

April 2013 alone. Another mosquito-borne disease which is prevalent in the country is malaria.

Malaria affects a total of 14 million people in the Philippines. An additional to these is

chikungunya which is a new viral infection caused by same species of mosquito that causes

dengue, the Aedes aegypti and the Aedes albopictus mosquitoes. Lastly, there is Lymphatic

filariasis, more commonly known as Elephantiasis which is a disease caused by an infection with

filarial worms which enter the human body through mosquito bites. (Know more about Mosquito-

borne diseases, 2013).

Mosquitoes are accountable for over 300 million clinical cases and one million deaths

yearly worldwide. Ineffective measures and the complexity of illness life cycles need integrated

approaches to control, eliminate, and eventually eradicate diseases (Molecular and population

Biology of Mosquitoes..., 2013).

Mosquitoes are tiny blood-sucking insects that depend on standing water to reproduce.

Female mosquitoes must feed on blood to lay eggs. They feed by sticking their mouthparts into the

skin of an animal and sucks blood rapidly. More often, they carry viruses that can be transmitted

to a person while they are feeding (Mosquito-Borne Diseases, 2013).

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Environmental conditions like temperature and moisture are where the length of

mosquito life cycle between species depends. Nonetheless, the life cycle of all mosquitoes consists

of the egg, larval, pupal and adult stages. Male mosquitoes feed on the nectar of the plants only

while females extract the blood of the hosts in order to develop and nourish eggs. Nearly all

mosquitoes lay their eggs directly into water and others lay their eggs just near but not within the

water. Within 24 to 48 hours, the eggs will hatch into larvae. The larvae will soon grow

approximately 5mm in length. They breathe through siphon or air tubes. The larger ones can be

seen floating just above the surface of waters that are infested. Within a span of seven to 10 days,

the larvae will enter the stage of a pupa. Pupae are also visible upon the surface of the breeding

site. When a mosquito is fully developed, it will emerge from its pupal case and will become a

mosquito. The new adult, at this time, will stand upon the water to dry its wings and prepare for its

flight. The female mosquitoes will then seek an animal to suck on and feed. They are capable of

flying for miles and lays over 100 eggs at a time. Larvae and pupae ordinarily cannot survive

without water so if a water source evaporates while they are still in these stages, they will die.

(Mosquito Life Cycle, 2013)

Several approaches were extensively used to control the threat posed by these

mosquitoes. Over the years, synthetic insecticides were introduced but although these are

effective, the insect tends to develop resistance to such products (Jirakanjanakit et al., 2007).

Aside from being costly, the use of these repellents also generates problems such as environmental

pollution and has toxic side effect to humans (Sarwar, Ahmad & Toufiq, 2009). This imposes a

need for other alternative methods which are efficient, economic and environmentally safe.

Botanical derivatives materialize as a possible larvicide for the Aedes aegypti or the common

household mosquito (Zhu et al., 2008).

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

The genus Bambusa (family Poaceae) consists of 120 bamboo species indigenous to

Asia and the New World. B. vulgaris (common bamboo) is a widely-grown species with several

infra-specific taxa, known variously as varieties or cultivars, including forms with variously green

and yellow-striped culms which are sometimes placed in distinct varieties or even species.

‘Wamin’ is a common cultivated form with ventricose to very short, concertina-like internodes

(Stapleton, 2007).

In this study, the larvicidal activity of BAMBOO (Bambusa vulgaris) against the larvae

of common species of mosquitoes found within the Philippines will be investigated.

1.2 Research Objectives

The general objective of the study is to determine the Larvicidal Activity of BAMBOO

(Bambusa vulgaris) leaves extract collected in New Leyte, Agusan del Sur as mosquito (Aedes

aegypti) larvae.

The following were the specific objectives of the study:

1. To test the Larvicidal activity of BAMBOO (Bambusa vulgaris) leaves extract against

mosquito (Aedes aegypti) larvae.

2. To determine the average mortality rate of mosquito larvae treated with Bamboo leaves

extract.

3. To determine if there is a significant difference between the different concentrations of the

BAMBOO (Bambusa vulgaris) leaves extract in killing mosquito (Aedes aegypti) larvae.

4. To determine if BAMBOO (Bambusa vulgaris) leaves extract is comparable to the

commercial larvicide.

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

1.3 Research Problem

In this regards, the researchers were determined to answer the following problems:

1. Does bamboo leaves extract have larvicidal activity against mosquito larvae.

2. What is the average mortality rate of mosquito larvae treated with bamboo leaves extract at

different concentrations?

3. Is there a significant difference on the mortality rates between the different concentrations of

bamboo leaves extract and the positive control?

1.4 Hypotheses

1. Bamboo (Bambusa vulgaris) leaves extract does not have larvicidal activity against mosquito

larvae.

2. There is no significant difference between the different concentration of Bambusa vulgaris

(BAMBOO) leaves extract in killing mosquito (Aedes aegypti) larvicide. Bambusa vulgaris

(BAMBOO) leaves extract is not comparable to the positive control against mosquito (Aedes

aegypti) larvae.

1.5 Conceptual Framework

Application and Mortality

Potential Mosquito
Bamboo leaf extract Of Mosquito (Aedes
Larvicide
aegypti) larvae

Figure 1.1 Conceptual Paradigm of the Study

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Figure 1 shows the main concept of this study which is to test the potency of BAMBOO

(Bambusa vulgaris) leaves extract as larvicide against the escalating cases of mosquito (Aedes

aegypti). Mainly, it serve as a natural source of larvicides to eventually reduce cases of dengue

cases that is rapid increasing problem in the Philippines.

The availability of the Bamboo leaves in the locality is very essential to make use of the

potential larvicides to kill mosquito larvae.

1.6 Significance of the Study

This study aims to discover the potential use of BAMBOO (Bambusa vulgaris) leaves

extract in eradicating mosquito (Aedes aegypti) larvae and as a substitute for commercial

pesticides.

The result of this study may help the people not only in Agusan del sur but in the whole

country in a way of new idea on preventing mosquitos to transmitted dangerous diseases. This

study may also help farmers to sell their bamboo plants and increase their income. However, this

study will give idea to the manufacturers to promote the use of non-harmful and organic substance

as an effective larvicide.

1.7 Scope and delimitation of the Study

This research will only focus on the BAMBOO (Bambusa vulgaris) leaves extract as a

potential (Aedes aegypti) larvicide. Laboratory experimentation and analysis is limited only to

Larvicidal property of this plant. The study will conducted at Agusan Sur National Science High

School, Science Laboratory room. This study will exceed more or less 24 hours. Three treatments

with different concentrations of Bamboo leaves extract were used and each treatment containing

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

three replicates with 10 larvae. The potentiality of the bamboo leaves extract only focused on

testing it to the Aedes aegypti mosquito larvae.

1.8 Definition of terms

The following terms are defined in order to give a clearer understanding on how the words are

used in study.

Aedes aegypti the yellow fever mosquito, is a mosquito that

can spread dengue fever, chikungunya, Zika

fever, Mayaro and yellow fever viruses, and

other disease agents.

Bamboo genus Bambusa (family Poaceae) consists of

120 bamboo species indigenous to Asia and

the New World. B. vulgaris (common

bamboo) is a widely-grown species with

several infra-specific taxa, known variously as

varieties or cultivars, including forms with

variously green and yellow-striped culms

which are sometimes placed in distinct

varieties or even species. ‘Wamin’ is a

common cultivated form with ventricose to

very short, concertina-like internodes

(Stapleton, 2007).

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Concentration it refers to the percentage by volume of bamboo

leaves extract in a solution and is described as

25%, 50%, 75% and 100%

Dengue is a mosquito-borne tropical disease caused by the

dengue virus.

Hematophagous it is the practice by certain animals of feeding on

blood

Larvicide it refers to a pesticide that kills mosquito larvae

Mosquito Larvae they refer to wrigglers obtained from the culture

of Mosquito on duration of five to seven days.

Potential It refers to the level of effectiveness of Bamboo

leaves extract killing mosquito larvae.

1.9 Review of Related Literature

Mosquitoes act as a vector for most of the life threatening diseases like malaria, yellow

fever, dengue fever, chikungunya ferver, filariasis, encephalitis, West Nile Virus infection, etc.

Under the Integrated Mosquito Management (IMM), emphasis was given on the application of

alternative strategies in mosquito control. The continuous application of synthetic insecticides

causes development of resistance in vector species, biological magnification of toxic substances

through the food chain and adverse effects on environmental quality and non target organisms

including human health. Application of active toxic agents from plant extracts as an alternative

mosquito control strategy was available from ancient times. These are non-toxic, easily available

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

at affordable prices, biodegradable and show broad-spectrum target-specific activities against

different species of vector mosquitoes.

Mosquitoes can transmit more diseases than any other group of arthropods and affect

million of people throughout the world. WHO has declared the mosquitoes as “public enemy

number one”1. Mosquito borne diseases are prevalent in more than 100 countries across the world,

infecting over 700,000,000 people every year globally and 40,000,000 of the Indian population.

They act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue

fever, chikungunya ferver, filariasis, encephalitis, West Nile virus infection, etc., in almost all

tropical and subtropical countries and many other parts of the world.

Mosquito larvae, called “wrigglers,” are aquatic, with a large head and thorax and

narrow, wormlike abdomen; they typically hang just below the water surface, breathing air

through tubes at the end of the abdomen. When disturbed, they wriggle downward.

The pupae, called “tumblers,” are curled like a comma and also hang just under the water

surface, breathing through air tubes.

Adult mosquitoes are small flies that look a lot like their cousins in the fly family, the

crane flies and midges. Female mosquitoes, however, drink blood from vertebrate animals.

Adults have one pair of transparent wings; upon close inspection, you can see a fringe of

hairs and scales along the edges and veins of the wings. The legs are long, and there is a long

proboscis (pro-BAH-siss) that is used like a straw for drinking. The antennae are featherlike in

males. (MDC Discover Nature)

All mosquitoes need standing or quiet water in which to lay their eggs. Adults of different

species have different habitats and active times. Some prevail near floodwaters or temporary

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

pools, others in houses, in irrigated pastures, marshes, or streams. Some species bite in the day,

while others are most active at dawn, dusk, or night. Eliminating standing water is an effective

way of reducing mosquitoes. Follow pesticide directions carefully. Educate yourself about the

many ways to manage mosquitoes.

After a blood meal, females rest a few days and develop 100-400 or more eggs. These

they usually deposit on the water, flying close and tapping the abdomen onto the surface. Eggs

hatch in a few days and spend about a week as “wrigglers.” The pupal stage lasts 2-3 days, after

which adults emerge, climbing out onto the water surface. Adults mate within a few days, and

females begin seeking blood. The life cycle usually takes a few weeks, but when conditions are

right, it can take only 10 days. (MDC Discover Nature)

Background Bamboo leaf extract solution (BLES) and sodium copper chlorophyllin

solution (SCCS) are known for their anti-oxidant activities. Oral malodor is often related with

periodontal pathogens. The present study was undertaken to investigate the anti-bacterial effect

of both BLES and SCCS on anaerobic periodontal bacteria producing oral malodorous volatile

sulfur compounds (VSC). Methods Porphyromonas gingivalis W83 (PG), Prevotella intermidai

TDC19B (PI), Fusobacterium nucleatum ATCC25586 (FN) and Prevotella nigrescence

ATCC33563 (PN) were investigated as oral isolated bacteria. VSC production ability of the oral

strains was investigated by gas chromatography. With serial dilution of BLES or SCCS, the

strains PG, PI, FN or PN were cultured anaerobically with AnaeroPack at 37 ℃ for 3 days. For

the determination of anti-bacterial action of BLES or SCCS, the inoculum was cultured with

original concentrations of BLES 0.16% (w/v) or SCCS 0.25% (w/v). Results Gas

chromatography exhibited that all strains, PG, PI, FN and PN were responsible for producing a

high range of H2S and a moderate range of CH3SH. Anti-bacterial effect of BLES or SCCS on

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

the strains was observed. Inhibition of BLES or SCCS on the strains was revealed as

concentration dependent. BLES or SCCS inhibited bacterial proliferation at higher

concentrations (PG; 0.04% BLES or 0.03% SCCS, PI; 0.002% BLES or 0.03% SCCS, FN;

0.005% BLES or 0.01% SCCS, PN; 0.01% BLES or 0.015% SCCS). No viable bacterial colony

observed at original concentration of BLES 0.16% or SCCS 0.25%. Strain growth was

eliminated from inhibition at lower concentrations (PG; 0.02% BLES or 0.015% SCCS, PI;

0.001% BLES or 0.015% SCCS, FN; 0.002% BLES or 0.007% SCCS, PN; 0.005% BLES or

0.007% SCCS). Conclusion High concentrations of both BLES (0.16%) and SCCS (0.25%)

show superior inhibiting capability on all four oral malodor associated periodontal anaerobes

during testing, suggesting that these compounds might have a beneficial effect. (bamboo leaf

extract_ Topics by Science.gov.mhtml)

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Chapter II

Methodology

Concentration Trial 1 Trial 2 Trial 3

AT MR AT MR AT MR
TREATMENT

% % %
100% Bamboo Leaves
T1 T1 T2 T2 T3 T3
Extract
75% Bamboo Leaves Extract T1 T1 T2 T2 T3 T3
50% Bamboo Leaves Extract T1 T1 T2 T2 T3 T3
Positive Control
T1 T1 T2 T2 T3 T3
(Victor WP)
2.1 Research Design

Table 1.1 Research Design

Legend: AT= After Treatment MR=Mortality Rate

T1, T2, and T3 = number of dead mosquito larvae.

Table 1.1 Research Design

Table 1.1 shows the research design of the study. There were 3 experimental treatments

Utilized, which were the Bamboo (Bambusa vulgaris) plant extract, Vistor WP serve as the

positive control and water as negative control. These treatments were used fro testing the

larvicidal effect of Bamboo (Bambusa vulgaris) leaves extract against mosquito (Aedes aegypti)

larvae on its mortality rate.

2.2 Materials

The materials and equipments will be used are:

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Mosquito Larvae Blender

Stirring Rod Petri Dish

Gloves Bucket

Strainer Plastic Containers (4)

Bamboo Leaves Beaker

2.3 Procedures

2.3.1 Collection and Extraction of Bamboo Leaves

Bamboo leaves were collected from P3-A, New, Leyte,

Awa, Prosperidad, Agusan del Sur, 500 grams of Bamboo

leaves were washed, chopped and blended. The extracts were

filtrated using a strainer.

2.3.2 Cultivation of Mosquito Larvae

The mosquito larvae were cultured for duration of 7-10

days. The bucket was filled with water and handfuls of manure

were added for mosquitoes to be feed on. The mosquito eggs

were checked frequently. The larvae were fed with larval food

(dog, biscuits and yeast in the ratio 3:1). After 7 days, the

larvae were taken from the jar and used as a subject in the

study.

2.3.3 Identification of Mosquito Larvae

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

The cultured mosquito will be brought to the Malaria Eliminating Hub, Patin-ay, Agusan

del Sur for determining of the type of this mosquito bioassay test.

2.3.4 Experimental Set-up

The concentration was

varied to 50% and 100% by volume respectively. There were three treatmens used. Treatment (1)

25%% concentration containing 20ml of extract and 30 ml distilled water. Treatment (2) 50%

concentration containing 50ml of extract with no distilled water. Treatment (3) 100%

concentration containing 70ml of extract with no distelled water.

2.3.4 Statistical Treatment

The Mortality data was analysed by using SPSS 16.0 software. Univariate Analysis of

Variance with Post Hoc Analysis was used for the scrutiny of the results.

2.3.5 Waste Disposal

The materials were washed after performing the application and extraction of Bamboo

leaves. Other important apparatus, equipment and hazardous materials which are beyond the

researcher’s handling concern must properly disposed carried out by the laboratory aide or

custodian. While the dead larvae was disposed using a needle.

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

2.4 Flowchart

COLLECTION AND EXTRACTION

OF BAMBOO LEAVES

CULTIVATION AND
IDENTIFICATION OF
MOSQUITO LARVAE

EXPERIMENTAL SET-UP

WASTE DISPOSAL

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Chapter III
Results and Discussion
Table 3.1 Experimental Set-up
Concentration Trial 1 Trial 2 Trial 3
AT MR AT MR AT MR
100% Bamboo Leaves 10 100.0000% 10 100.00% 10 100.0000%
TREATMENT

Extract
50% Bamboo Leaves 10 86.6667% 10 86.6667% 10 86.6667%

Extract
25% Bamboo Leaves 10 76.6667% 10 76.6667% 10 76.6667%
Extract
Positive Control 10 100.0000% 10 100.0000% 10 100.0000%
(Victor WP)

Legend: AT= After Treatment MR=Mortality Rate

T1, T2, and T3 = number of dead mosquito larvae.

Number of Dead Mosquito Larvae

Mortality Rate= ×100
Number of Mosquito Larvae Tested

Table 3.1 Experimental Set-up

The table 3.1 shows that the overall data collected after the experimentations. The

mortality rates between the different concentrations of the treatments were computed and also

the average mean of mortality rates were obtained. For Bamboo (Bambusa vulgaris) leaves

extract, it has the mean average of 100.000% of mortality rate.

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Table 3.2 Univariate Analysis of Variance of the Treatment of Bamboo (Bambusa vulgaris)
leaves extract
Mortality_Rate
Type III Sum of
Table 3.3 Post Hoc Analysis
Subset
Source Squares df Mean Square F Sig.
Treatment a N 1 2 3
Corrected Model 1068.668 3 356.223 3.817E5 .000
Intercept 25% concentration
87988.031 3 76.6467
1 87988.031 9.427E7 .000
Treatment 50% concentration
1068.668 3 3 86.6467
356.223 3.817E5 .000
Error Victor WP (+) .007 3 7 1.0000E2
.001
Total 100% 90147.076 11
2 1.0000E2
Corrected Total concentration 1068.674 10
a. R SquaredSig. 1.000
= 1.000 (Adjusted R Squared = 1.000) 1.000 1.000
Means for groups in homogeneous subsets are displayed.

Table 3.2 Univariate Analysis of Variance of the Treatment of

Bamboo (Bambusa vulgaris) leaves extract

Table 3.2 shows that the computed significant value of the extract is .000 which is less

than 0.05 hence, the null hypothesis which states that there is no significant difference on the

Larvicidal activity of Bamboo (Bambusa vulgaris) against mosquito (Aedes aegypti) larvae will

be rejected. This implies that there is significant difference between the different concentrations

of the Bamboo (Bambusa vulgaris) leaves extract treated on mosquito (Aedes aegypti) larvae.

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Table 3.3 Post Hoc Analysis of the Treatments of Bamboo (Bambusa vulgaris), Positive

Control (Vistor WP), and the Negative Control against Mosquito Larvae

Table 3.3 shows the comparison of Bamboo (Bambusa vulgaris) leaves extract together

with the positive and negative control in killing mosquito (Aedes aegypti). The Bamboo

(Bambusa vulgaris) leaves extract has an percent mean mortality on mosquito (Aedes aegypti) of

100.00% which is comparable to the positive control Victor WP with an percent mean mortality

on mosquito (Aedes aegypti) of 100.00%. Both of the Bamboo leaves and the positive control are

highly toxic to mosquito larvae which are found in the same subset to the positive control.

This implies that the Bamboo (Bambusa vulgaris) and Victor WP as positive control are

both could highly kill a mosquito larvae wheras to the negative control.

Chapter IV

Summary of findings, Conclusions and Recommendations

4.1 Summary of Findings

This study investigates the Larvicidal activity of Bamboo (Bambusa vulgaris) leave extract

against the mosquito (Aedes aegypti) larvae.

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

Getting the Mortality Rate and Univariate Analysis of Variance was used for the

determination of the significant difference of the Bamboo (Bambusa vulgaris) leave extracts as

Mosquito Larvicide on Aedes aegypti. While Post Hoc Analysis was used to test the significant

difference between the different concentrations of Bamboo (Bambusa vulgaris) leaves extract in

killing mosquito (Aedes aegypti) larvae which is found to be comparable to the Victor WP as the

positive control. Overall, the scrutiny of the results was all done by the SPSS 16.0 software.

Results showed that the Bamboo (Bambusa vulgaris) is highly toxic to mosquito larvae

and it shows that there is significant difference between the different concentration of Bamboo

(Bambusa vulgaris) leaves extract in killing on mosquito (Aedes aegypti) larvae. This means that

plant extract was found to have a Larvicidal Potency against Aedes aegypti mosquito larvae.

4.2 Conclusions

This study concluded that Bamboo leave extract effective as larvicides against Aedes

aegypti.

The following conclusion were deduced:

1. There is a potential Larvicidal activity in Bamboo (Bambusa vulgaris) leaves extract

against mosquito (Aedes aegypti) larvicide.

2. The Bamboo(Bambusa vulgaris) leaves extract percent mean mortality is comparable to

the positive control (Victor WP) against mosquito (Aedes aegypti) larvae.

3. There is a significant difference between the different concentrations of Bamboo

(Bambusa vulgaris) leaves extract in killing mosquito (Aedes aegypti) larvae.

4.3 Recommendations

1. Extraction of crude extract using Rotary Evaporator is highly recommended.

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 Bamboo (Bambusa vulgaris) as Larvicidal against
Mosquito (Aedes aegypti) Larvae

2. Since it is a potent larvicide, it is highly recommended especially on wet places prone

to mosquitos.

3. The waste of Bamboo leaves extract can be used as a main subject for a construction

paper.

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