UNIVERSITI TEKNOLOGI MARA PERAK

KAMPUS TAPAH
FACULTY OF APPLIED SCIENCES
BIOLOGICAL TECHNIQUES AND SKILLS
BIO300
PROJECT PROPOSAL
AS1205F
TITLE:
EFFICIENCY OF CALENDULA sp.
EXTRACT (Diptera: CULICIDAE)
REPELLENT

Page | 1

PART A:
i.

RESEARCHER BACKGROUND

PROJECT LEADER/ MATRIC NO. /CONTACT NO.:

NURUL NABIHA MOHD ROSEDI

ii.

PROJECT MEMBERS:

NO.

NAME

01

FATIN FATINAH MOHD

NORAIZAN
NUR FARAH AZREN MOHD
NAZERI
NUR HAZWANI RAZAK

02
03

iii.

MATRIC
NUMBER

CONTACT
NUMBER

PROJECT SUPERVISOR:
MADAM NADIA NISHA BT. HAJI MUSA

iv.

CO-SUPERVISOR (if any)

MADAM NUNSHAIMAH SALLEH

v.

LECTURER (Lecture/Laboratory)
MADAM MARLINA BINTI MOHD MYDIN

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PART B: RESEARCH PROJECT PROPOSAL

i.

OBJECTIVES:
To determine the efficiency of calendula as repellent to culicidae

ii.

Hypothesis

Null : There is no repellent efficiency of calendula on culicidae .

Alternative : There is repellent efficiency of calendula on culicidae

iii.

Variable
1. Independent variable : Part of calendula.
2. Dependent variable : The diversity of culicidae after using the
calendula as repellent.
3. Control variable : Time taken to observe the result.
Sampling technique.
Concentration of extract.

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iv.

SIGNIFICANCE OF PROJECT:

This project was presenting the new problem solving to repel the common
insect that regularly disturb our living home, mosquitoes. Commonly the repellent
product that have been produced was use ingredients base on calendula sp and also
only non-organic chemical. Now, was presenting the innovation of mosquitoes
repellent that use flowers as the base ingredients Calendula sp., or the local name is
Marigold flower was find out have a chance to repel the mosquitoes.

The main extraction methods, namely Rotavapor method, were investigated

using the chosen raw material. In evaluating the extraction methods, some important
factors affecting the extraction process were investigated. These include the standing
or exposure time, solids to solvent ratio and temperature. The effect of these
parameters on the concentration of the extracted lutein, and the rate of extraction were
examined. In this study, it was found that rotavapor method of extractions, acetone
yielded the highest amount of lutein compared to other solvents used in the study. In
general, the amount of lutein extracted was proportional to the standing time and the
raw materials to solvent ratio. In all the operating conditions, maximum achievable
lutein concentration was present, after which a plateau condition was observed. The
maximum achievable lutein concentration was dependent on the operating conditions.

A study on the extraction of lutein from selected locally grown vegetation

was carried out as the first step in developing the most suitable method of producing
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the lutein. The active component in Marigold flower can be used as repellent same
effective as DEET and at the same time can lower the cost of mosquitoes repellent.
This repellent is non-toxic and very safe for every age group and do not harm pets
also. The smell of the oil relieves from new mosquitoes repellent can calms the body.

We are using marigold in this experiments as repellent because it is cheap

and easy to found. Besides that, we are not using any harmful chemical that give bad
effect to environment.

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v.

DEFINITION OF TERMS/CONCEPTS:

Terms
Calendula

Definition
A plant that is grown for its bright yellow
or orange flowers, which is one of the
richest natural sources of xanthophylls,

Culicidae

mainly lutein
A slender long-legged fly with aquatic
larvae.The bite of the bloodsucking female
can transmit a number of serious diseases

Aqueous

including malaria and encephalitis.

Aqueous is a term used to describe a

FTIR

system which involves water.

FT-IR stands for Fourier Transform
InfraRed, the preferred method of infrared

Rotavapour

spectroscopy
A piece of apparatus consisting of a motor
unit that rotates the evaporation flask, a
vacuum system, a heated water bath and a
condenser , which is used to remove
solvents from samples under reduced

Lutein

pressure
A deep yellow pigment of the xanthophyll
class, found in the petal of plants, in egg

Bare leg

yolk, and in the corpus luteum

By using bare leg will attract the
mosquitoes which can collect to calculate
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their population.

vi.

LITERATURE REVIEW:

1. Culicidae

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The scientific classification of culicidae start with Kingdom is Animalia, Phylum

is Arthropoda, Class is Insecta, Order is Diptera, Suborder is Nematocera,
Infraorder is Culicomorpha, Superfamily is Culicoidea.

Source : http://www.wumcd.org/mosquito/lifecycle.gif

1.1 Culicidaes Life Cycle

There are some 3300 species of mosquitoes belonging to 41 genera, all contained
in the family Culicidae. This family is divided into three subfamilies: Toxorhynchitinae,
Anophelinae (anophelines) and Culicinae (culicines).
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According to Laurel(2014), the mosquito goes through four separate and distinct
stages of its life cycle which is egg, larva, pupa and adult. Eggs are laid one at a time or
attached together to form "rafts" and float at the surface of water. The larva lives in the
water and comes to the surface to breath . Larvae shed (molt) their skins four time,
growing larger after each molt. Most larvae have siphon tubes for breathing and hang
upside down from the water surface. The pupa stage is resting, non-feeding stage of
development, but pupae are mobile, responding to light changes and moving (tumble)
with a flip of their tails toward the bottom or protective areas. This is the time the
mosquito change to adult. The newly emerged adult rests on the surface of the water for a
short time to allow itself to dry and all its body part to harden. The wings have to spread
out and and dry properly before it can fly.

According to Amrita University(2013), mosquito must have water in which to

complete their life cycle. A female mosquito can produce 500 eggs before she dies. The
length of life of adult mosquito depends on factors like temperature, humidity, sex of the
mosquito.

1.2: Mosquitoes characteristics

According to Jessia (2005), a winged termite travelling in swarm will have a

small, black, ant-like head with two with wings that give a slightly violet sheen in the
light. Department of Entomology Texas A&M University (2010), states that winged

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Formosan termite reproductive or swarmers are yellowish-brown and 12-15mm

(0.5-0.6 inch) in length. According to Dawn (2001) states that the primary
reproductive also called swarmers or alates, vary in body colour from dark brown to
black.

1.3: Mosquitoes lifestyle

According to Leisnham PT, LaDeau SL, Juliano SA (2014). Aedes albopictus

would show a greater increase in the proportion of sites occupied and per site
abundance during the rainy season than Aedes aegypti, due to its high dry season egg
mortality and heavy rainy season competitive superiority. While the proportion of
occupied sites increased from early to late rainy season for Aedes albopictus. They
actually decreased for Aedes aegypti, suggesting that rainy-season competition from
Aedes albopictus had a particularly severe negative effect on the distribution of Aedes
aegypti. Numerous studies have shown that the seasonal patterns of Aedes aegypti
and Aedes albopictus abundances are linked with local rainfall, and these patterns
have been interpreted as being a result of dry-season egg mortality and rainy-season
competitive ability.
Culex is more sensitive to dry conditions than are Aedes and experience high
dry-season mortality resulting in low abundance early in the rainy season, and that
abundances would increase during the rainy season as more water filled container
habitats become available as stated by Leisnham PT, LaDeau SL, Juliano SA (2014).
The adult females bite people and animals throughout the night, indoors and outdoors.

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During the day they are inactive and are often found resting in dark corners of rooms,
shelters and culverts. Numerous studies have shown that the seasonal patterns of
Aedes aegypti and Aedes albopictus abundances are linked with local rainfall, and
these patterns have been interpreted as being a result of dry-season egg mortality and
rainy-season competitive ability.

For mansonia, this species is very sensitive to low humidities and most males
and unfed females are dead after exposure to sulphuric acid and sodium hydroxide
solutions also at a temperature of 27-29C as stated by B. R. Lacresce (n.d).

Anopheles mosquitos are active between sunset and sunrise. Each species has
specific peak biting hours, and there are also variations in their preference for biting
indoors or outdoors. The anophelines that enter houses to feed often rest indoors for a
few hours after feeding. They may then leave for outdoor sheltered resting sites,
among them vegetation, rodent burrows, cracks and crevices in trees or in the ground,
caves and the undersides of bridges. Alternatively, they may stay indoors for the
whole period needed to digest the blood-meal and produce eggs. Indoor resting is
most common in dry or windy areas where safe outdoor resting sites are scarce.

1.4: Mosquitoes Habitat

According to Mosquito World (2015), mosquitoes can live in almost any

environment, with the exception of extreme cold weather. They favor forests, marshes,
tall grasses and weeds, and ground that is wet. Mosquito live in floodwater habitat
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include drainage ditches that fill during storm, woodland pools created by melting snow,
or spring and early summer rains, floodplains along the banks of stream and rivers, also
irrigated pastures and field.

According to University of Florida (n.d), mosquito habitat divided by two .

Floodwater mosquito and permanent water mosquito. Floodwater mosquito live in moist
area that will eventually dry out and the mosquito eggs also dried and become encased in
the cracks and crevices of the dried mud. This species need water to lay their eggs and the
eggs need to dry out before they can hatch. There are estimate that mosquito that live in
floodwater habitat between 0.7 and 1.3 million per acre. While permanent water mosquito
cannot withstand drying out. Water is is necessary for a complete life cycle. This
mosquito live in the place that permanently have water without drying .

According to Orkin (2015), hot, humid environment are most amenable to mosquito
growth and survival. Infestation can occur easily in tropical areas. Mosquito larvae are
active in transient water such as flood water, ditches and woodland pools. Mosquito
prefer stagnant water within which they lay egg such as pond, marshes and swamps.

2. Natural Plant

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Source: hdimgegallery.net

2.1 Plant Description

Marigolds are most useful in repelling or warning away insects when

planted along with vegetables and fruits. It has pungent smell that repels insects
including mosquitoes and usually the villagers planted it around their house and
farm. According to Noor Asikin , (2012) this statement is supported by Vasudevan
(1997) and also by Sarin (2004). It also is a potential plant whose essential oil from
flowers has been effective repellent against insects.

According to the RHS Gardening, Calendula derived from the Latin

calendae , the first day of the month on which interest paid, in reference to the long
period of bloom (2008, as cited by Eileen).Calendula has been used for medical
purpose in the treatment of jaundice and as a substitute for saffron in the treatment of
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measles and smallpox. Marigold is beneficial for the eyes, and old herbal reports.

2.2 Active Compound in Marigold Flower

A number of papers are now available on the repellent activities from

Marigold against different type of mosquito species. It has an active ingredient which
is lutein. Lutein is one type of material that can be use in repellent to replace DEET.
It obtained from the Marigold flower petals where it is one of the major constituent
of yellow or orange fruits and vegetables such as mango, papaya, prunes and others.

Lutein is usually useful for preventing cataract and arteriosclerosis,

enhances immunity and also has significant functions for preventing cancer
formation where it can delay cancer development. The functional group of lutein is
xanthophyll. This statement is according to Noor Asikin, (2012),supported by Liu &
Fan, (2010). Figure 2.1 shows the lutein structure with molecular formula is C 40H56O2
and molecular weight is 568.87 g/mol. It is insoluble in water, but soluble in fats and
lipophilic solvents.

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Figure 2.1 : Lutein structure

According to Lim, Serena (2003) there was two main extraction methods, which
are conventional method and rotavapor method, were investigated using the chosen
raw material. Behr Labor Technique (n.d) states that continue the heat solvent vessel
until all the solvent has been evaporated and condensed in the rotavapor extractor.
According to The Interactive Lab Primer (n.d), when a compound of low solubility
needs to be extracted from solid mixture at rotavapor interaction can be carried out.
The technique places a specialized piece of glassware in flask and a condenser.
According to Asikin (2012), the content of lutein extracted from Marigold flower
was found to be 2.5354% and the maximum amount of lutein extracted was obtained
at temperature 45C, volume of solvent at 150 ml and extraction time at 30 minutes.
The optimum results demonstrated that temperature was influenced variable on the
extraction content of lutein. The extraction rate constant, k of lutein decreased with
increasing temperature and volume of solvent, and the k values were (0.0405-0.2712)
min-1. According to Hojnik et al. (2008, as cited at Sakinah,2012) was find that when
Page | 15

temperature is increase from 20C to 40C, a small increase in the final extraction
efficiency of lutein can be observed and remains constant with further rise of
temperature to 60C. According to Chromatoqr (2013), the addition of acetone
enhance the extraction yield of lutein in many flowers..

According to Javier(1988), was developed a new reversed-phase HPLC

method for separating in a single step lutein and the different lutein fatty acid esters
in colour sources and in other coloured products. The commonest commercial source
of lutein is the flower of the marigold plant, where it is found esterified with one or
two fatty acids and constituents about 90%(w/) of the petals. Lutein and lutein fatty
acids ester content in two different parts (petals and calyces) of flower-head from
different types of marigold. (1998, as cited at Roberta).

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3.1: FTIR Test

According to Thermo Nicolet corporation (2001), FTIR are stand for

Transform Infrared, the suggested method of infrared spectroscopy that passes
through sample. Nancy(2015) states that FTIR spectrometers usually used in organic
synthesis, polymer science, petro chemical engineering, pharmaceutical industry and
food analysis. According to Magni`(1999), the combination of IR absorption
spectroscopy and mass spectrometry allows the chemical composition and structure
of the most relevant plasma-produced neutral species, the ionic plasma composition
and also the chemical composition of the nanometer-sized particles to be precisely
identify. Jonas (2015) states that FTIR can investigate the gas phase during the
chemical vapour deposition of silicon carbide from CH3SiCl3. Andrew (2015) states
that FTIR spectroscopy imaging can determine the concentration in ibuprofen in
PEG at which dimerization of ibuprofen can be avoided. FTIR spectra can be analyze
to access the molecular state and degree of polymer.

According to Yan(2011), stated that Fourier transform infared (FTIR)

spectroscopy was used for obtaining vibrational spectra of the Tagetes erect a petal
and Chrysanthemum petal. The content of lutein esters in the petal was studied by
analysis of the relative absorption intensity.

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Source: Instrument Laboratory

3.2 Rotavapor

According to Department of chemistry Wake Forest University(n.d), the purpose

of rotavapor is to remove low boiling organic chemical, usually solvents from a
mixture of a compound. The solvents are removed by a simple distillation. The
rotavapor is design under vacuum and to heat the sample at the same time. A cold
finger is used to condense the vapors to a liquid, which are trapped in a separate
flask. According to University of Toronto Scarborough (2010), an important
concept that this technique applies is that liquid boil when the vapor pressure is
equal to external pressure or atmospheric pressure. The machine utilizes a lower
pressure than atmospheric pressure which allows solvents to boil at lower
temperature. Other than that , The rotation increase the surface area and therefore
evaporation proceeds more rapidly.

Page | 18

Source : Instrument Laboratory

The extraction efficiency of marigold yellow pigment by different extraction

methods were presented according to Sheng(2001).The stability of this pigment to
light and temperature was also studied. The results showed that the extraction
efficiency of marigold pigment was lvigh to 6,21mg/L by the combined extractant of
hexane, acetone, and methanol. Additionally, marigold yellow pigment was sensitive
to light but insensitive to temperature.

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4.3 Bare Leg

According to Renaud(2005), the increased attractiveness was not due to an

intrinsic attractiveness of gametocytes carriers but to the infection status associated
with the presence of gametocytes. The parasites change the infected individuals
breath or body odour, as these are involved in attracting mosquitoes at the distances.
While transpiration and body temperature also attract mosquitoes at these distances,
these factors are less likely to be involved in the manipulation as the infection was
asymptomatic in all of the children involved in the study.

According to Minhas S , Sekhon H (20130), human bare-leg catches

(landing catches) of Aedes adults or indoor resting collections of adults are most
commonly used to assess adult Aedes populations. The data collected is used to
calculate the number of Aedes mosquitoes landing / biting on a single human bait per
hour (number per man hour). The survey must assess the mosquito population over a
large area; therefore the collectors should move from house to house, not lingering in
one place. In the same way, indoor resting collections can be made.

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4.4 Data analysis

According to Kent State University(n.d), SPSS is a user friendly program

that facilitates data management and statistical analysis. Creating data on SPSS is
enter the variables and value manually into SPSS to create a new dataset. The
variables and values comes from file or table that have already prepared. Variables
are the key part of all data sets. A variable is name, type, label, or other attributes.
According to Institute Digital Research and Education(2015), creating standardized
variables in SPSS is very simple to get the mean, siginificant value and other simple
test statistic.

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vii.

RESEARCH METHOD

1. Collect 300g of flowers without roots.

Series no:UiTM/P8A2/A/1W823/2015W00010
MODEL:PERKIN ELMER/FRONTIER

2.Do the FTIR test to check the presence of lutein in the petals
2.Click icon on desktop 'spectrum'.
3.Log in
4.Insert basic parameter instrument
5.Then do the background scan
6.Scan the sampel
7.After that, print out the spectrum result.
8.Log out.
9.Separate the petals from the sepals
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10.Dry the flower for 3 days under the sunlight

11.Soak the dry flower in the acetone for one week
12.Set the apparatus as shown

ID NO:109162 Rotavapour r-3(Bucchi)

13.Fill 3/4 of water in the heating bath

14.Insert 1/4 of the flower that have been soak in the acetone in the evaporating
flask. Then, connect the conical flask to the vapour duct, then fall off the
metal bracelet and turn the combi-clip clockwise.
15.Turn on the main switch
16.Open the tap
17.Turn on the ON/OFF switch. Then set the up down button to 40-50 degree
Celcius
18.Let off the brake lift. Turn the rotation knob and set the turning speed to (1-

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10)
19.Turn on the vacuum controller. Press the button menu and set.
20.Turn the selection knob and set the pressure at 556 atm for acetone sample.
21.Press ok and the button start.
22.Wait until all the acetone evaporate then, turn the rotation knob and stop the
evaporating flask.
23.Press the stop button twice to let out the pressure
24.Switch off heating bath and the vacuum controller
25.Press the lift brake in the holder to lift up the evaporating flask
26.Take the sample in the evaporating flask. Clean up the apparatus.
27. Keep the extraction oil in the glass bottle to avoid evaporate and durable
28. By using bare legs method which take 5 days to collect the population of the
mosquitoes with lutein and without lutein for 5 days
Day 1
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 2
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 3
1.30pm-7.30pm
17.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 4
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm

Name
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Page | 24

Day 5
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm

Fatin and Nabiha

Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani

29. By following shift time which 1.30 p.m until 7.30 p.m and 7.30 p,m to 1.30
a,m to 7.30 am the mosquitoes will collect along the time scheduled.
30. Repeat procedure 29 with lutein for 5 days (2/9/2015-6/9/2015) and without
lutein for 5 days (7/9/2015-11/9/2015)
31. The mosquitoes collected will be kept in bijou bottle.
32.The collected mosquitoes is calculated by using SPSS to get standard
deviation, mean and significance
33.Record all the results in the table

viii.

LIST OF EQUIPMENTS/FACILITIES

1) Rotavapour
2) FTIR
3) Test tube
4) Beaker
5)Measuring cylinder
6)Bijou bottle
7)Spray bottle
8)Compound Microscope

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LIST OF CHEMICALS/MEDIA:
1)Lutein(in the petals)
3)500 ml of Acetone

ix.

GANTT CHART

Research Activities /
Duration
Discussion

June

July

August

September

W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4 W1 W2 W3 W4
with

Advisor
Submission of first

draft
Proposal

Submission
Proposal

Presentation

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Collection

of

participants

information
Interpretation

of

data and result

Thesis writing
Submission of final
project
Final

Project

presentation

x.

FLOW CHART

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5. Extract the
sample using
rotavapour.

6. Spray
the
extraction
oil at the
leg

4. Soak in the
acetone solvents for
one week.

Collect the samples

without the roots.

3. Separate all the

petals from sepals.

2. Do the FTIR test

using raw samples.

Page | 28

Fresh Marigold

Series no: UiTM/P8A2/A/1W823/2015W00010

MODEL: PERKIN ELMER/FRONTIER

Page | 29

Dried Marigold Flower

Soaked Marigold Flower

Page | 30

ID NO:109162 Rotavapour r-3(Bucchi)

Bare Leg
Technique

Calculation
Page | 31

xi.

REFERENCES

Noor Asikin Ahmad Sfri (2012), Study on Extraction Kinetics and Formation of
Natural Mosquito Repellent Solution from Marigold Flower Extract: Universiti
Malaysia Pahang

Eartheasy.com(2001),5 Easy to Grow Mosquito-Repelling Plant at

http://eartheasy.com/blog/2011/04/5-easy-to-grow-mosquito-repelling-plants

Wikepidia (n.d). Marigold at https://en.m.wikepidia.org/wiki/marigold

S.Miroslav, K.Stanislav, F.Miroslav, K.Borivoj & K-vlastiml (2014).Lutein

Content In Marigold Flower at
http://www.agriculturejournals.cz/publicfiles/138021.pdf

Dr.P.May, Dr.K.W.Quinn, Flaver Naturextra.Kte GmBh (2014), Supercritical

Marigold FlowerCO2-Extract at
http://www.flavex.com/fileadmin/flavex.de/user_upload/veroeffentlichungen/engl
ish/suprcritical_marigold_flower_co2-extract-cst2014.pdf

Flower Expert (2005). Marigold- at

Page | 32

http://theflowerexpert.com/content/growingflowers/flowersandseasons/marigolds

R.James (n.d), Journal of Microbiology and Biology education, Retrieved from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004744/

Univertsity California chemwiki (n.d), How An FTIR Spectrometers Operates.

Retrieved from
http://chemwiki.ucdavis.edu/Physical_chemistry/spectroscopy/Vibrational_spectr
oscopy/How_an_FTIR_spectrometer_Operates

Thermo.Nicolet.Corporation(2001) .Thermo Nicoletto Fourier Transform

Infared.SpectrometryThermo Nicolet .Retrieved from
mmrc.caltech.edu>FTIR>FTIR intro

Jonason.D (2014), Surveys Moths Using Light Traps: Effect Of Weather And Time
Of Year .Retrieved from http://journals.plos.org/plosone/article?
id=10.1371/journal.pone.0092453

A.G.Douglas, (2005), Standard Deviation and Stardard Error . Retrieved from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255808/

Page | 33

Lauurence (n.d), The Biology Of Two Species Of Mosquito, Mansonia Africana

(THEOBALD) And Mansonia Uniformis (THEOBALD), Belonging To The Subgenus
Mansoniaoides (DIPTERA, CULICIDAE). Retrieved from

http://www.mosquitocatalog.org/files/pdfs/075600-0.pdf

Mosquitos and other biting Diptera .Retrieved from

http://www.who.int/water_sanitation_health/resources/vector007to28.pdf

Minhas S , Sekhon H (2013), Entomological Survey For Dengue Vector:

INTERNATIONAL JOURNAL OF MEDICAL AND APPLIED SCIENCES.

Retrieved from http://earthjournals.org/ijmas_258.pdf

Ralph Harbach (2009), Mosquito Taxonomic Inventory. Retrieved from

http://mosquito-taxanomic-inventory.info/itemculexltemgt-classification

Centers for disease control and prevention (n.d). Retrived from

anopheles/www.cdc.gov/malaria/about/biology/mosquitoes

Mosquito Taxonomic Inventory (2008). Retrieved from http://mosquitotaxonomic-inventory.info/simpletaxonomy/term/6218

Page | 34

Kyle Harfma (1999), Aedes Albopictus. Retrieved from

http://animaldiversity.org/accounts/aedes-albopictus/

Elsevier B.V. (1991), Journal of Chromatography. Retrieved from

www.sciencedirect.com/science/article/pii/S0021967300942663

Roberta P. (1998), Abstract. Retrieved from

www.sciencedirect.com/science/article/pii/s092666909710005X

Yan.C.Q (2011), Identification of three species of chrysanthemum petal by fourier

transform infared spectroscopy. Retrieved from en,cnki,com.cn/article-en/CJFD
TOTAL-GPSS201103131.htm

Medical Entomology, Westmead Hospital (1996), Aedes Aegyapti. Retrieved from

medent.usyd.edu.au/photos/aedes/.20aegyapti.htm

Mount. L(2014). Mosquito Life cycle. Retrieved from : www.mosquito.org/lifecycle

Amrita.olabs.co.in(2013). Life Cycle of Mosquito. Retrieved from :

amrita.olabs.co.in/?sub=79&brch=15&sim=138&cnt=1

Mosquito World(2015). Your Guide To Effective Mosquito Control. Retrieved

Page | 35

from : www.mosquitoworld.net/about-mosquitoes/habitats/

Florida Medical Entomology Laboratory(n.d), Mosquito habitat. Retrieved from :

mosquito.ifas.ufl.edu/Mosquito_Habitats.htm

Orkin,LLC(2015). Mosquito Habitat. Retrieved from :

www.orkin.com/other/mosquitoes/moaquito-habitats/

Page | 36

DECLARATION AND SIGNATURE

We are hereby declaring that the project is based on our original

work except for quotations and citations which have been duly
acknowledge. We are conducting our investigations with honesty
and integrity

SIGNATURE OF PROJECT LEADER: ________________________

( NURUL NABIHA BINTI MOHD
ROSEDI )
SIGNATURE OF PROJECT MEMBER:
FATIN FATINAH BINTI MOHD NORAIZAN:
NUR HAZWANI BINTI RAZAK :
NUR FARAH AZREN BINTI MOHD NAZERI:

Signature of Supervisor: _________________________________

Received date: ____________________________

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Returned date: ____________________________

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